A Pretargeted Imaging Strategy for EGFR-Positive Colorectal Carcinoma via Modulation of Tz-Radioligand Pharmacokinetics.

Purpose: Previously, we successfully developed a pretargeted imaging strategy (atezolizumab-TCO/[Tc]HYNIC-PEG-Tz) for evaluating programmed cell death ligand-1 (PD-L1) expression in xenograft mice. However, the surplus unclicked [Tc]HYNIC-PEG-Tz is cleared somewhat sluggishly through the intestines, which is not ideal for colorectal cancer (CRC) imaging. To shift the excretion of the Tz-radioligand to the renal system, we developed a novel Tz-radioligand by adding a polypeptide linker between HYNIC and PEG.

Procedures: Pretargeted molecular probes [Tc]HYNIC-polypeptide-PEG-Tz and cetuximab-TCO were synthesized. [Tc]HYNIC-polypeptide-PEG-Tz was evaluated for in vitro stability and in vivo blood pharmacokinetics. In vitro ligation reactivity of [Tc]HYNIC-polypeptide-PEG-Tz towards cetuximab-TCO was also tested. Biodistribution assay and imaging of [Tc]HYNIC-polypeptide-PEG-Tz were performed to observe its excretion pathway. Pretargeted biodistribution was measured at three different accumulation intervals to determine the optimal pretargeted interval time. Pretargeted (cetuximab-TCO 48 h/[Tc]HYNIC-PEG-Tz 6 h) and (cetuximab-TCO 48 h/[Tc]HYNIC-Polypeptide-PEG-Tz 6 h) imagings were compared to examine the effect of the excretion pathway on tumor imaging.

Results: [Tc]HYNIC-polypeptide-PEG-Tz showed favorable in vitro stability and rapid blood clearance in mice. SEC-HPLC revealed almost complete reaction between cetuximab-TCO and [Tc]HYNIC-polypeptide-PEG-Tz in vitro, with the 8:1 Tz-to-mAb reaction providing a conversion yield of 87.83 ± 3.27 %. Biodistribution and imaging analyses showed that the Tz-radioligand was cleared through the kidneys. After 24, 48, and 72 h of accumulation in HCT116 tumor, the tumor-to-blood ratio of cetuximab-TCO was 0.83 ± 0.13, 1.40 ± 0.31, and 1.15 ± 0.21, respectively. Both pretargeted (cetuximab-TCO 48 h/[Tc]HYNIC-PEG-Tz 6 h) and (cetuximab-TCO 48 h/[Tc]HYNIC-polypeptide-PEG-Tz 6 h) clearly delineated HCT116 tumor. Pretargeted imaging strategy using cetuximab-TCO/[Tc]HYNIC-polypeptide-PEG-Tz could be used for diagnosing CRC, as the surplus unclicked [Tc]HYNIC-polypeptide-PEG-Tz was cleared through the urinary system, leading to low abdominal uptake background.

Conclusion: Our novel pretargeted imaging strategy (cetuximab-TCO/[Tc]HYNIC-polypeptide-PEG-Tz) was useful for imaging CRC, broadening the application scope of pretargeted imaging strategy. The pretargeted imaging strategy clearly delineated HCT116 tumor, showing that its use could be extended to selection of internalizing antibodies.