Click-to-Release: Cleavable Radioimmunoimaging with [⁸⁹Zr]Zr-DFO-<i>Trans</i>-Cyclooctene-Trastuzumab Increases Tumor-to-Blood Ratio

One of the main challenges of PET imaging with ⁸⁹Zr-labeled monoclonal antibodies (mAbs) remains the long blood circulation of the radiolabeled mAbs, leading to high background signals, decreasing image quality. To overcome this limitation, here we report the use of a bioorthogonal linker cleavage approach (click-to-release chemistry) to selectively liberate [⁸⁹Zr]Zr-DFO from <i>trans</i>-cyclooctene-functionalized trastuzumab (TCO-Tmab) in blood, following the administration of a tetrazine compound (trigger) in BT-474 tumor-bearing mice. <b>Methods:</b> We created a series of TCO-DFO constructs and evaluated their performance in [⁸⁹Zr]Zr-DFO release from Tmab <i>in vitro</i> using different trigger compounds. The <i>in vivo</i> behavior of the best performing [⁸⁹Zr]Zr-TCO-Tmab was studied in healthy mice first to determine the optimal dose of the trigger. To find the optimal time for the trigger administration, the rate of [⁸⁹Zr]Zr-TCO-Tmab internalization was studied in BT-474 cancer cells. Finally, the trigger was administered 6 h or 24 h after [⁸⁹Zr]Zr-TCO-Tmab- administration in tumor-bearing mice to liberate the [⁸⁹Zr]Zr-DFO fragment. PET scans were obtained of tumor-bearing mice that received the trigger 6 h post-[⁸⁹Zr]Zr-TCO-Tmab administration. <b>Results:</b> The [⁸⁹Zr]Zr-TCO-Tmab and trigger pair with the best <i>in vivo</i> properties exhibited 83% release in 50% mouse plasma. In tumor-bearing mice the tumor-blood ratios were markedly increased from 1.0 ± 0.4 to 2.3 ± 0.6 (p = 0.0057) and from 2.5 ± 0.7 to 6.6 ± 0.9 (p < 0.0001) when the trigger was administered at 6 h and 24 h post-mAb, respectively. Same day PET imaging clearly showed uptake in the tumor combined with a strongly reduced background due to the fast clearance of the released [⁸⁹Zr]Zr-DFO-containing fragment from the circulation through the kidneys. <b>Conclusions:</b> This is the first demonstration of the use of <i>trans</i>-cyclooctene-tetrazine click-to-release chemistry to release a radioactive chelator from a mAb in mice to increase tumor-to-blood ratios. Our results suggest that click-cleavable radioimmunoimaging may allow for substantially shorter intervals in PET imaging with full mAbs, reducing radiation doses and potentially even enabling same day imaging.