Info
Background:
Reducing total-body radiation dose in macromolecule-based nuclear medicine remains a key challenge, particularly due to the prolonged circulation time of directly radiolabeled monoclonal antibodies (mAbs), which increases off-target radiation. Pretargeting strategies offer a promising solution by decoupling the targeting and radiolabeling steps, allowing clearance of unbound mAbs before delivering the radioactive payload.
Hypothesis/Objective:
Our lab previously reported a CB7-adamantane “host–guest” platform that demonstrated high tumor uptake and significantly reduced systemic radiation exposure, providing a modular pretargeting strategy. Building on this, we present a novel guest scaffold, nanodiamond (ND), with Ka values two orders of magnitude higher than adamantane (~1015 M⁻¹ vs ~1017 M⁻¹) and improved pretargeting performance. ND’s higher binding affinity and favorable pharmacologic profile yield exceptional tumor uptake and retention, making it this the most promising class of host:guest pretargeting radioligands to date.
Methods:
Guest radioligand 64Cu-NOTA-PEG2-DiDa, bearing a PEG2 linker and NOTA chelator, was synthesized and radiolabeled. In vitro characterization included determination of partition coefficient (log D), plasma stability, plasma protein binding, and blood half-life. The previously reported CB7-M5A (~1.8 CB7:M5A conjugation ratio) was prepared for pretargeting experiments and immunoreactivity confirmed to be >95% via cell binding assays with BxPC3 cells. The radioligand was then evaluated in vivo. Four cohorts of female nude mice bearing BxPC3 (CEA+) tumors (n=4/cohort) received CB7-M5A (100 µg in 250 µL PBS, pH 7.4) followed 6 days later by administration of 64Cu-NOTA-PEG2-DiDa (1.5 nmol, 7.4–8.8 MBq in 250 µL PBS, pH 7.4). Biodistribution was performed at 2, 4, 12, 24, and 48 h after radioligand injection. Small-animal PET/CT imaging was conducted at 4, 12, and 24 h, followed by ex vivo biodistribution. Urine stability was assessed using samples collected at 2 and 24 h post-injection. Control groups, assessed at 24 h post radioligand injection, received either excess cold Cu-NOTA-PEG2-DiDa (10 nmols/ mouse) 4 h prior 64Cu-NOTA-PEG2-DiDa administration or were administered radioligand only without CB7-M5A. The serial biodistribution data was used for estimated human dosimetry (70 kg adult male) calculations with OLINDA.
Results:
64Cu-NOTA-PEG2-DiDa was synthesized with a high radiochemical yield (>97%) and remained >94% intact in human plasma at 24 h. Hydrophilicity (log D₇.₄) was determined to be -1.07 ± 0.23 and the weighted blood half-life exhibited relative blood clearance of the radioligand at t½ ≈ 14 minutes. Radio-HPLC of urine samples collected 2 h after injection showed fully intact radioligand (>99 %) and no free 64Cu, indicating the ligand’s in vivo stability. Pretargeting experiments in BxPC3 xenograft-bearing mice using a 6-day lag time demonstrated rapid tumor accumulation and favorable clearance profile for the novel ND based radioligand. Biodistribution data showed tumor accumulation as early as 4 h (%ID/g = 13), with sustained tumor uptake through 24 h (%ID/g = 25). Pretargeted PET imaging showed rapid tumor contrast that was sustained across all time points. The radioligand cleared rapidly from blood and kidneys, reducing off-target exposure by 24 h. Blocking studies with excess cold ligand 1 and control studies with no prior antibody administration reduced tumor uptake to ≤ 0.5 %ID/g, confirming specificity. The dosimetry data corroborated the biodistribution and PET imaging results, confirming that the ND-based guest ligand scaffold has improved pretargeting properties compared to previously reported ferrocene and adamantane scaffolds.
Conclusion:
The CB7-DiDa platform establishes a proof of concept for an entirely new class of guest scaffold for pretargeting. It shows strong potential for pretargeted PET imaging achieving ~25 %ID/g tumor uptake at 24 h, combining stability, specificity, and low systemic radiation exposure. Ongoing studies exploring linker length effects and other chelator-radionuclide combinations represent the next steps of platform development.
Author
Anwesha Sil
Stony Brook Medicine