Development of a Novel Prosthetic Click-Linker for Radioiodination of Antibody-Based Radiopharmaceuticals with High Stability and Specificity

Radioiodine has been exploited in nuclear medicine for diagnostic and therapeutic purposes in various diseases. There are two radioiodination methods for biomolecules, that is, (1) direct radioiodination of tyrosine or histidine residue in a biomolecule and (2) indirect radioiodination by using a prosthetic group, which bridges radioiodine and the biomolecule. While directly radioiodinated biomolecules suffer from deiodination <i>in vivo</i>, the most commonly used indirect labeling method based on <i>N</i>-succinimidyl-3-[*I]iodobenzoate has a problem of inconvenience due to an high-performance liquid chromatography (HPLC) purification process. To tackle both issues, a novel prosthetic click-linker-antibody conjugate (3-[^123/125I]iodobenzoyl-PEG₄-tetrazine-TCO-PEG₄-trastuzumab (3-[^123/125I]IBTTT)) with favorable radiochemical yield (>57%) and purity (>99%) was developed using a fluorous tin-based organotin precursor with streamlined purification process utilizing fluorous solid-phase extraction (FSPE) cartridge and spin column. <i>In vitro</i> binding studies demonstrated that 3-[¹²⁵I]IBTTT maintained its biological activity with a <i>K</i>_D value (5.606 nM) comparable to that of unmodified trastuzumab (5.0 nM). <i>In vivo</i> imaging of 3-[¹²³I]IBTTT in a human epidermal growth factor receptor 2 (HER2)-expressing gastric cancer mouse model revealed favorable tumor accumulation and negligible thyroid uptake compared to directly radioiodinated trastuzumab ([¹²³I]trastuzumab). It was also confirmed, by blocking experiments and a biodistribution study, that the tumor accumulation of 3-[¹²³I]IBTTT was attributed to HER2-specific binding. In summary, we developed a novel radioiodinated prosthetic click-linker agent (3-[^123/125I]IBTTT) with favorable radiochemical yield, purity, stability, and <i>in vivo</i> behavior, providing a highly promising tool for targeted imaging and potential therapy of HER2-positive cancers.