Development of a Novel Prosthetic Click-Linker for\nRadioiodination of Antibody-Based Radiopharmaceuticals with High Stability\nand Specificity

Radioiodine has been\nexploited in nuclear medicine for diagnostic\nand therapeutic purposes in various diseases. There are two radioiodination\nmethods for biomolecules, that is, (1) direct radioiodination of tyrosine\nor histidine residue in a biomolecule and (2) indirect radioiodination\nby using a prosthetic group, which bridges radioiodine and the biomolecule.\nWhile directly radioiodinated biomolecules suffer from deiodination <i>in vivo</i>, the most commonly used indirect labeling method\nbased on <i>N</i>-succinimidyl-3-[*I]iodobenzoate has a\nproblem of inconvenience due to an high-performance liquid chromatography\n(HPLC) purification process. To tackle both issues, a novel prosthetic\nclick-linker-antibody conjugate (3-[<sup>123/125</sup>I]iodobenzoyl-PEG<sub>4</sub>-tetrazine-TCO-PEG<sub>4</sub>-trastuzumab (3-[<sup>123/125</sup>I]IBTTT)) with favorable radiochemical yield (>57%) and purity\n(>99%)\nwas developed using a fluorous tin-based organotin precursor with\nstreamlined purification process utilizing fluorous solid-phase extraction\n(FSPE) cartridge and spin column. <i>In vitro</i> binding\nstudies demonstrated that 3-[<sup>125</sup>I]IBTTT maintained its\nbiological activity with a <i>K</i><sub>D</sub> value (5.606\nnM) comparable to that of unmodified trastuzumab (5.0 nM). <i>In vivo</i> imaging of 3-[<sup>123</sup>I]IBTTT in a human epidermal\ngrowth factor receptor 2 (HER2)-expressing gastric cancer mouse model\nrevealed favorable tumor accumulation and negligible thyroid uptake\ncompared to directly radioiodinated trastuzumab ([<sup>123</sup>I]trastuzumab).\nIt was also confirmed, by blocking experiments and a biodistribution\nstudy, that the tumor accumulation of 3-[<sup>123</sup>I]IBTTT was\nattributed to HER2-specific binding. In summary, we developed a novel\nradioiodinated prosthetic click-linker agent (3-[<sup>123/125</sup>I]IBTTT) with favorable radiochemical yield, purity, stability, and <i>in vivo</i> behavior, providing a highly promising tool for\ntargeted imaging and potential therapy of HER2-positive cancers.