Development of a New Folate-Derived Ga-68-Based PET Imaging Agent

Abstract

Purpose The folate receptor (FR) has emerged as an interesting diagnostic and therapeutic drug target with many potential applications in oncologic and inflammatory disorders. It was therefore the aim of this study to develop a folate-derived Ga-68-based positron emission tomography (PET) imaging tracer that is straightforward to radiolabel and could be broadly used in clinical studies. We validated its target binding affinity and specificity and compared it to [99mTc]EC20, the folate single-photon emission computed tomography (SPECT) imaging tracer that has been most extensively studied clinically so far. Procedures The new folic acid-derived PET imaging agent is linked via a polyethyleneglycol linker to the chelator 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA). This new compound, NOTA-folate, was labeled with gallium-68. We tested the probe’s stability in human plasma and its selectivity in vitro, using the FR-positive KB cell line as well as the FR-negative A549 cell line. The pharmacokinetic profile of [68Ga]NOTA-folate was evaluated in FR-positive KB mouse xenografts. Following intravenous injection of [68Ga]NOTA-folate (383 ± 53 µCi), PET/computed tomography (CT) imaging studies as well as biodistribution studies were performed using KB tumor-bearing mice (n = 3). In vitro as well as in vivo studies were performed in parallel with the SPECT imaging tracer [99mTc]EC20. Results In comparison to [99mTc]EC20 (radiochemical yield (RCY) = 82.0 ± 2.9 %, 91.8 ± 2.0 % purity), similar radiochemical yield (87.2 ± 6.9 %) and radiochemical purity (95.6 ± 1.8 %) could be achieved for [68Ga]NOTA-folate. For both tracers, we observed high affinity for FR-positive cells in vitro and high plasma stability. In PET/CT and biodistribution studies, [68Ga]NOTA-folate appeared to display slightly superior in vivo performance in comparison to [99mTc]EC20. In detail, 68Ga-NOTA-folate showed very good tumor uptake and retention (6.6 ± 1.1 %ID/g), relatively low kidney uptake (21.7 ± 1.1 %ID/g), and very low liver uptake (0.38 ± 0.08 %ID/g). In vivo blocking studies using a fivefold excess of EC20 reduced the tumor uptake to 2.5 ± 0.7 %ID/g, confirming receptor specific binding of [68Ga]NOTA-folate in vivo. Conclusion We validated a new Ga-68 folate-based PET imaging agent with excellent pharmacokinetics and tumor uptake. Based on a head-to-head comparison between both tracers, [68Ga]NOTA-folate is a suitable imaging probe for the delineation of FR-positive tumors and a promising candidate for clinical translation.

Radiation dosimetry of 18F-AzaFol: A first in-human use of a folate receptor PET tracer

“…However, Etarfolatide is a single-photon emission tomography (SPECT) tracer with limited imaging resolution and inferior quantification compared to positron emission tomography (PET). In pre-clinical studies, folatederived 68 Ga-based PET probe has shown slightly superior in vivo performance in comparison to Etarfolide [9]. This compound is to the best of our knowledge under investigation in humans in an inflammatory indication (NCT03494114) and currently not published in a peerreviewed journal.…”

Leveraging PET to image folate receptor α therapy of an antibody-drug conjugate

“…Furthermore, an in vitro binding assay was performed with cell lines expressing different levels of FRα, as previously described [ 20 ]. One day prior to the in vitro experiment, the cells lines KB, OV90, and H2110, as well as A549 (each 1.0 × 10 6 cells per well, > 90% viability for each cell line) were seeded into 6-well plates containing growth medium (2.0 mL) and incubated at 37 °C to form subconfluent cell monolayers.…”

“…All in vivo experiments were carried out in female, athymic nude mice (Envigo; outbread; 6–8 weeks, 20–25 g). Subcutaneous KB and OV90 xenografts (150 ± 20 mm 3 ) were established as previously described [ 5 , 20 ]. In detail, suspended KB cells (1.5 × 10 6 , viability > 93%) or suspended OV90 cells (1.0 × 10 7 , viability: > 95%) in a solution containing a 1:1 mixture of Matrigel (Becton Dickinson, Bedford, MA) and cell culture media (no FBS for OV90 xenografts) were subcutaneously inoculated on the right shoulder of anesthetized mice (1.5–2.0% isoflurane (Baxter Healthcare) in medical air (2 L/min)).…”

Prospective of68Ga Radionuclide Contribution to the Development of Imaging Agents for Infection and Inflammation

“…The majority of the nuclear imaging agents based on folic acid or pteroic acid [ 155 ] were developed for diagnosis of cancers overexpressing FR receptors such as breast, cervical, ovarian, colorectal, nasopharyngeal, renal, and endometrial cancers. Various 68 Ga-labelled agents demonstrated accumulation in cell cultures and mice bearing folate-receptor positive human nasopharyngeal carcinoma cell line (KB) xenografts [ 6 , 156 – 162 ]. [ 68 Ga]Ga-DOTA-PEG-FA comprising folic acid was investigated for the detection and quantification of inflammatory response to medical implants using mice with subcutaneously implanted polylactic acid and poly(N-isopropylacrylamide) particles as a model [ 60 ].…”