Synthesis, preclinical evaluation, and clinical translation of [68Ga]Ga-Asp2-JR11, a SSTR2 antagonist for PET imaging of neuroendocrine neoplasms.

Abstract

Purpose: Somatostatin receptor subtype 2 (SSTR2) is overexpressed in well-differentiated neuroendocrine neoplasms (NENs) and serves as a key target for positron emission tomography (PET) imaging. While SSTR2 agonists such as [⁶⁸Ga]Ga-DOTA-TATE are widely used clinically, recent evidence suggests that antagonist radioligands can bind more receptor sites without inducing internalization, potentially offering superior imaging performance. Here, we report the synthesis, preclinical validation, and pilot clinical translation of [⁶⁸Ga]Ga-Asp₂-JR11, a novel SSTR2 antagonist radioligand featuring an -Asp₂-PEG₂- linker designed to enhance hydrophilicity and receptor engagement for PET Imaging of NENs.

Methods: Asp₂-JR11 was synthesized by modifying the NOTA-JR11 backbone, and its binding properties were evaluated via molecular docking, in vitro assays, and in vivo imaging. Radiolabeling with ⁶⁸Ga was performed for Asp₂-JR11, NOTA-JR11, and DOTA-TATE. We conducted cell uptake, internalization, PET/CT imaging, biodistribution, and blocking studies in AR42J (SSTR2-positive) and HCT116 (SSTR2-negative) tumor models. A first-in-human study included nine patients with NENs who underwent [⁶⁸Ga]Ga-Asp₂-JR11 PET/CT and [¹⁸F]FDG PET/CT imaging, along with dosimetry assessment.

Results: Docking analysis showed that Asp₂-JR11 maintained equivalent binding energy to NOTA-JR11 but formed more hydrogen bonds with SSTR2 (10 vs. 5), suggesting enhanced stability. [⁶⁸Ga]Ga-Asp₂-JR11 demonstrated high radiochemical purity (> 95%), higher molar activity (12.9-14.8 GBq/µmol), and greater hydrophilicity (LogD = - 3.18 ± 0.01) than comparators. In AR42J cells, [⁶⁸Ga]Ga-Asp₂-JR11 exhibited rapid uptake (9.95 ± 0.10%AD/10⁶ cells at 30 min) and low internalization (17.63 ± 0.91% at 120 min), with significantly higher uptake than [⁶⁸Ga]Ga-DOTA-TATE and [⁶⁸Ga]Ga-NOTA-JR11 in both in vitro and micro PET/CT studies (e.g., 10.67 ± 0.16 vs. 7.79 ± 0.50%ID/g at 30 min, p < 0.05). In vivo imaging and biodistribution confirmed higher tumor-to-background ratios and reduced off-target organ uptake, notably in the kidneys, pancreas, and spleen. Tumor uptake was significantly inhibited by co-injection of SSTR2 ligands, confirming specificity. In human subjects, [⁶⁸Ga]Ga-Asp₂-JR11 showed favorable biodistribution and rapid clearance via renal excretion, with the spleen showing the highest transient uptake. Tumors were clearly visualized as early as 12 min post-injection and maintained strong contrast up to 120 min. Dosimetry revealed the highest absorbed dose in the urinary bladder wall (5.78 × 10⁻² mSv/MBq), with an effective whole-body dose of 9.94 × 10⁻³ mSv/MBq. Comparative PET/CT imaging in nine patients (33 lesions) showed that [⁶⁸Ga]Ga-Asp₂-JR11 detected 21 lesions missed by [¹⁸F]FDG, particularly in the liver. For liver metastases, [⁶⁸Ga]Ga-Asp₂-JR11 yielded higher SUV_max (8.8 vs. 3.2, p = 0.001) and tumor-to-background ratio (6.9 vs. 2.7, p < 0.0001) compared to [¹⁸F]FDG.

Conclusion: [⁶⁸Ga]Ga-Asp₂-JR11, a novel SSTR2 antagonist PET probe, exhibits improved hydrophilicity, receptor engagement, tumor uptake, and in vivo stability compared to existing agents. Its high tumor-to-background contrast and favorable dosimetry profile underscore its potential as a clinically translatable radioligand for sensitive and specific PET imaging of NENs.