Name: Automated Preparation of 68Ga Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment (Video) | JoVE Journal
Uploaded: 2025-04-25T14:50:00.000+00:00
Duration: 10 min 33 s
Description: Learn automated synthesis of Gallium 68 3BP3940 for PET imaging of the tumor microenvironment using a GAIA V2 module. This 20-minute GMP-compliant protocol enables efficient radiopharmaceutical production.

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The authors thank Yasmine Soualy, St&#233;phane Renaud and &#201;lodie Gaven for their help in preparing the radiolabeling reactions presented in this manuscript.

The authors have no commercial partnerships or funding sources that would result in a real or perceived conflict of interest relating to this work to disclose.

This work presents a GMP-compliant automated preparation protocol for the synthesis of [68Ga]Ga-3BP-3940 using a GAIA module and a GALLIAD generator. This method was adapted from protocols used in our center for gallium-68 radiolabeling of vectors such as PSMA ligands44 and other FAP inhibitors43,46 for clinical PET imaging, with slight modifications.
The production process was designed to be simple a...

In recent years, targeting the tumor microenvironment (TME) has attracted considerable interest in diagnostic and therapeutic applications1. The abundance of cell types, signaling molecules, and extracellular matrix (ECM) macromolecules within the TME offers a wide range of potential molecular targets2. Among the resident and infiltrating host cells, cancer-associated fibroblasts (CAFs) form a distinct subset of fibroblasts within the TME, phenotypically different from normal fibroblasts. CAFs play crucial roles in tumor progression, metastasis, immune evasion, and therapy resistance through unique cellular and molecular characteristics3. These mesenchymal cells exhibit an activated phenotype marked by the expression of fibroblast activation protein (FAP). Molecularly, CAFs secrete a complex array of cytokines, chemokines, growth factors (e.g., TGF-β, IL-6, and CXCL12), and ECM proteins (e.g., collagen, fibronectin), which remodel the ECM and foster a pro-tumorigenic environment4.
As a highly specific protein that is overexpressed and localized on the extracellular surface of the CAF membrane, FAP displays all the characteristics of a reliable molecular target, especially for nuclear medicine and radiopharmaceutical applications5. In this context, quinoline-based small molecule inhibitors of FAP (FAPI), functionalized with a DOTA group, were developed and quickly introduced into clinical use6,7,8. Specifically, FAPI-04 and FAPI-46 radiolabeled with gallium-68 (β+ emitter, t1/2 = 68 min) for positron emission tomography (PET) imaging have demonstrated significant value in fibrotic diseases, cardiology, and oncology8,9, particularly for cancers where [18F]fluorodeoxyglucose ([18F]FDG) has limited utility10. However, while their contributions to oncology and nonmalignant diseases imaging are undeniable, small molecule FAPIs exhibit certain limitations for targeted radionuclide therapy (TRT) applications, particularly due to their suboptimal intratumoral residence time, which can lead to unintended irradiation of healthy tissue11. To address this issue, several strategies have been explored, such as the design of multivalent ligands11,12 or the use of therapeutic radionuclides with short half-lives13,14,15. New molecular scaffolds with a high affinity for FAP and triggering a high proportion of cell internalization have also been developed.
One of these is the pseudopeptide derivative FAP-2286. It contains a 7-amino acid sequence, cyclized and linked to a DOTA chelator by a 1,3,5-benzenetrimethanethiol moiety16. An initial study in humans demonstrated that [68Ga]Ga-FAP-2286 exhibits a biodistribution profile similar to [68Ga]Ga-FAPI-46, with slightly higher physiological uptake in the liver, kidneys, and heart17. In this study, 64 patients, primarily with cancers of the neck, liver, stomach, pancreas, ovaries, and esophagus, underwent PET imaging with [68Ga]Ga-FAP-2286 for cancer staging or detection of recurrence: uptake of [68Ga]Ga-FAP-2286 was notably higher than [18F]FDG in primary tumors, lymph node metastases, and distant metastases, enhancing image contrast and lesion detectability. All primary tumors were visible with [68Ga]Ga-FAP-2286 PET/CT, whereas [18F]FDG PET/CT missed almost 20% of the lesions. For involved lymph nodes, detection rates were higher with [68Ga]Ga-FAP-2286, as well as for bone and visceral metastases. Another study in a smaller group of 21 patients with a variety of cancer diseases also demonstrated the excellent sensitivity of this imaging agent, reflecting the diagnostic efficiency of [68Ga]Ga-FAP-228618. More specific studies have focused on a single type of cancer, such as urothelial or lung cancer, highlighting once again the high potential of [68Ga]Ga-FAP-2286 for clinical molecular imaging4,5. Regarding therapy, a preliminary study investigated the use of FAP-2286 radiolabeled with lutetium-177 (β- emitter, t1/2 = 6.7 d) in 11 patients with diverse progressive, metastatic cancers19. Most patients received two treatment cycles spaced 8 weeks apart, and the average administered dose per cycle was 5.8 ± 2.0 GBq of [177Lu]Lu-FAP-2286. The drug demonstrated prolonged intratumoral retention, with an effective half-life of approximately 44 h in bone metastases. Given the acceptable side effects, these findings paved the way for larger-scale clinical trials: the safety and efficacy of [177Lu]Lu-FAP-2286 are currently being assessed in the phase 1/2 LuMIERE clinical trial, sponsored by Novartis (NCT04939610)7,8. Further smaller-scale research protocols are documented in the literature9,20, and multiple case reports have been published21,22,23,24,25,26, demonstrating the efficacy and excellent tolerability of this TRT.
Minimal structure modifications made on FAP-2286 led to the optimized analog 3BP-3940 (Figure 1)27. Although scientific literature on this vector molecule remains limited, early studies have been conducted for both imaging and therapeutic applications. A preliminary report describes the use of [68Ga]Ga-3BP-3940 in 18 patients with various end-stage metastatic carcinomas and concludes that this radiopharmaceutical is a suitable PET imaging agent, emphasizing its excellent tumor-to-background ratio and very low kidney uptake28. In another work, a single pancreatic cancer patient with liver metastases received 150 MBq of [68Ga]Ga-3BP-3940 for PET imaging, which demonstrated intense uptake in the primary tumor and metastatic lesions29. The same patient subsequently received a single dose of 9.7 GBq of [177Lu]Lu-3BP-3940 for TRT. The treatment was well tolerated, with no significant changes in vital signs or biological parameters. A different study presented the initial human results of a theranostic approach using 3BP-3940: patients were selected with [68Ga]Ga-3BP-3940 PET imaging and then received 3BP-3940 labeled with different isotopes (177Lu, 90Y, or 225Ac), administered alone or in tandem combinations (e.g., 177Lu + 225Ac) in 1-5 treatment cycles30. Outcomes included one complete remission, four partial remissions, three stable diseases, and 12 disease progressions. The cohort's (n = 28) median overall survival was 9 months from the start of TRT.
<img alt="Chemical structure, 68Ga radiolabeled compound, organic molecule diagram, nuclear medicine." class="xfigimg" src="/files/ftp_upload/68356/68356fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Chemical structure of [68Ga]Ga-3BP-3940. <a href="https://www.jove.com/files/ftp_upload/68356/68356fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The 68Ga radiolabeling process for experimental radiopharmaceuticals such as FAP-2286 and 3BP-3940 generally involves a synthesis module to automate the preparation step. Notably, method automation ensures process robustness and GMP compliance and minimizes operator radiation exposure in comparison with manual preparation methods31,32,33. In many cases, such a protocol is expected by regulatory authorities as a part of an investigational medicinal product dossier (IMPD) before authorizing a center to manufacture the corresponding experimental radiopharmaceutical34. To date, very little detailed information on the automated 68Ga radiolabeling of anti-FAP pseudopeptides is available in the literature29,35,36,37,38. Moreover, the data reported generally applies only to a given model of synthesizer. The type of 68Ga generator used can also bring certain specificities, as the different commercially available solutions are characterized by specific volumes of 68Ga3+ eluate in HCl (usually 0.1 M), which can have a direct impact on automated radiolabeling conditions.
In this context, we present a detailed protocol for the rapid and efficient automated radiolabeling of the pseudopeptide 3BP-3940 with 68Ga, using the GAIA V2 synthesis module. This synthesizer relies on the use of a tubing set comprising three ramps of five manifolds each, connected to a peristaltic pump to control fluid flow. It also features a vial oven for reaction medium heating, several radioactivity probes, and a pressure sensor to monitor these parameters within the system. Although not as widespread as some other models, this automaton is used routinely in our center and is installed in a growing number of facilities31,39,40,41,42,43,44. A GALLIAD 68Ge/68Ga generator was used in this work without prepurification of the 68Ga eluate. This method is designed to offer a robust, fast, and convenient solution for the production of [68Ga]Ga-3BP-3940, also optimizing radiation protection for operators during radiolabeling. This is also the first preparation protocol for this radiopharmaceutical to be reported on this specific synthesizer model, and in such detail.

<table><tbody><tr><td>0.2 &amp;micro; filters</td><td>VWR</td><td>514-0515</td><td>For filtration of buffer and antioxidant solutions and final radiolabeling product</td></tr><tr><td>Acetonitrile for HPLC</td><td>Sigma Aldrich</td><td>34851-2.5L</td><td>For HPLC control of radiochemical purity</td></tr><tr><td>Ammonium acetate</td><td>Sigma Aldrich</td><td>238074</td><td>For the preparation of one of the mobile phases for TLC control</td></tr><tr><td>C18 column for HPLC</td><td>VWR</td><td>EQV-3C18-1503</td><td>For HPLC control of radiochemical purity</td></tr><tr><td>Calibrated dose calibrator (CRC25)</td><td>Capintec</td><td>-</td><td>For measuring the radioactivity of the final product and the various components of the module post-synthesis</td></tr><tr><td>Citrate buffer solution, pH 4</td><td>Thermofisher</td><td>258585000</td><td>Mobile phase for TLC controls</td></tr><tr><td>Eppendorf tube 5 mL Biopur</td><td>Sigma Aldrich</td><td>EP0030119479</td><td>For the preparation of buffer and antioxidant solutions</td></tr><tr><td>Extension line (30 cm)</td><td>Vygon</td><td>1159.03</td><td>For the connection of the generator to the tubing set</td></tr><tr><td>Gallium-68 generator</td><td>IRE Elit</td><td>-</td><td>For in situ generation of [68Ga]gallium chloride</td></tr><tr><td>Gamma counter (Hidex AMG)</td><td>Hidex</td><td>-</td><td>For half-life and radiochemical purity assessment</td></tr><tr><td>HPLC station</td><td>Shimadzu</td><td>-</td><td>For HPLC control of radiochemical purity</td></tr><tr><td>iTLC-SG plates</td><td>Agilent</td><td>SGI0001</td><td>For TLC control of radiochemical purity</td></tr><tr><td>L-methionine</td><td>AppliChem</td><td>A1340</td><td>For antioxidant solution preparation</td></tr><tr><td>Male/male adapter</td><td>Vygon</td><td>893.00</td><td>For the connection of the generator to the tubing set</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>320390-1L</td><td>For the preparation of one of the mobile phases for TLC control</td></tr><tr><td>Needles (21G, Sterican)</td><td>B Braun</td><td>4657543B</td><td>For solution transfers prior to radiolabeling</td></tr><tr><td>pH paper</td><td>VWR</td><td>85409.600</td><td>To test the pH of the radiolabelling product</td></tr><tr><td>Pipette 1000 &amp;micro;L (Gilson PIPETMAN)</td><td>Fisher Scientific</td><td>12346132-1000</td><td>For precise liquid measurement and transfer</td></tr><tr><td>Pipette 200 &amp;micro;L (Gilson PIPETMAN)</td><td>Fisher Scientific</td><td>12326132-200</td><td>For precise liquid measurement and transfer</td></tr><tr><td>Pipette Tips, 100-1000 &amp;mu;L</td><td>Charles River</td><td>D1000IW</td><td>For precise liquid measurement and transfer</td></tr><tr><td>Pipette Tips, 2-200 &amp;mu;L</td><td>Charles River</td><td>D200IW</td><td>For precise liquid measurement and transfer</td></tr><tr><td>Radiochromatograph</td><td>Elysia-Raytest</td><td>-</td><td>For TLC control of radiochemical purity</td></tr><tr><td>Radiosensor for HPLC</td><td>Elysia-Raytest</td><td>-</td><td>For HPLC control of radiochemical purity</td></tr><tr><td>Reagents kit</td><td>ABX</td><td>RT-101</td><td>Provides ethanol 60%, NaCl 0.9%, WFI bag, C18 cartridge, 0.2 &amp;micro; terminal filter, aeration needles, terminal needle and waste vial</td></tr><tr><td>Shielded container</td><td>LemerPax</td><td></td><td>For radiation attenuation of the radiolabeling product</td></tr><tr><td>Single-use plastic spatula</td><td>Corning</td><td>3005</td><td>For the preparation of reagents</td></tr><tr><td>Sodium acetate trihydrate EMPROVE</td><td>Sigma Aldrich</td><td>1.28204</td><td>For reaction buffer preparation</td></tr><tr><td>Sterile sealed vials (glass type 1)</td><td>Curium</td><td>TC-ELU-5</td><td>For final conditioning of buffer, antioxidant and radiolabeling solutions</td></tr><tr><td>Sterile tubing set</td><td>ABX</td><td>RT-01-H</td><td>For automated synthesis of [68Ga]Ga-3BP-3940</td></tr><tr><td>Sterile water for irrigation</td><td>B Braun</td><td>0082479E</td><td>For the preparation of one of the mobile phases for TLC control</td></tr><tr><td>Synthesis module (GAIA)</td><td>Elysia-Raytest</td><td>-</td><td>For automated synthesis of [68Ga]Ga-3BP-3940</td></tr><tr><td>Syringe (1 mL, low dead-volume)</td><td>B Braun</td><td>9166017V</td><td>For peptide in buffer conditionning and addition of methionine in NaCl 0.9%</td></tr><tr><td>Syringes (10 mL)</td><td>Becton Dickinson</td><td>309649</td><td>For methionine in NaCl 0.9% and conditionning</td></tr><tr><td>Syringes (3 mL)</td><td>Becton Dickinson</td><td>309658</td><td>For methionine and ethanol 60% conditionning</td></tr><tr><td>TLC migration tanks</td><td>Fisher Scientific</td><td>50-212-281</td><td>For TLC control of radiochemical purity</td></tr><tr><td>Trifluoroacetic acid (suitable for HPLC)</td><td>Sigma Aldrich</td><td>302031-100ML</td><td>For HPLC control of radiochemical purity</td></tr><tr><td>Tubes for gamma counter</td><td>-</td><td>-</td><td>For half-life and radiochemical purity assays preparation</td></tr><tr><td>Ultrasonic bath</td><td>Selecta</td><td>3000683</td><td>For sonication of prepared solutions</td></tr><tr><td>Vector molecule (3BP-3940)</td><td>MedChemExpress</td><td>HY-P10131&amp;nbsp;</td><td>Vector molecule to be radiolabeled</td></tr><tr><td>Vial for HPLC with glass insert</td><td>Sigma Aldrich</td><td>29385-U and SU860066</td><td>For HPLC control of radiochemical purity</td></tr><tr><td>Vortex mixer</td><td>VWR</td><td>444-5900P</td><td>For stirring the prepared solutions</td></tr><tr><td>Water for HPLC</td><td>Sigma Aldrich</td><td>34877-2.5L-M</td><td>For HPLC control of radiochemical purity</td></tr><tr><td>Water for injection, 10 mL flasks</td><td>Aguettan</td><td>34009 370 641 0 1</td><td>For solutions preparation</td></tr></tbody></table>

automated preparation of [68ga]ga-3bp-3940 on a synthesis module for pet imaging of the tumor microenvironment

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

The synthesis process developed on the GAIA module allows fast 68Ga radiolabeling of 3BP-3940 in 21-22 min. This protocol was designed to work with pharmaceutical grade 68Ge/68Ga generator GALLIAD, which produces 1.1 mL of 68Ga eluate in 0.1 M HCl. The volume and molarity of the reaction buffer were finely tuned according to this amount of acid to obtain a reaction pH between 3.5 and 4, necessary for optimal radiolabeling45. Thus, sodium acetate with a f...

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

automated-preparation-68gaga-3bp-3940-on-synthesis-module-for-pet

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Learn automated synthesis of Gallium 68 3BP3940 for PET imaging of the tumor microenvironment using a GAIA V2 module. This 20-minute GMP-compliant protocol enables efficient radiopharmaceutical production.

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