DOTA-tetrazine probes with modified linkers for tumor pretargeting

Introduction: Pretargeted radioimmunoimaging and -therapy approaches building on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have yielded impressive results in recent years and have proven a vital alternative to biological pretargeting systems. After improvement of the TCO-antibody conjugates, we here report on our evaluation of a new series of radiolabeled Tz-probes.

Methods: Four new Tz-probes were synthesized, radiolabeled with lutetium-177, and characterized in vitro in terms of lipophilicity, reactivity, and stability in PBS and mouse serum. The in vivo biodistribution profile and tumor-targeting potential of the probes were evaluated in LS174T tumor-bearing mice pretargeted with TCO-antibody conjugates using non-pretargeted mice as control.

Results: Radiolabeling of all probes proceeded in high yields providing the ¹⁷⁷Lu-labeled tetrazines in >95% radiochemical purity without any further purification. In mouse serum, half-lives of the probes varied between 8 and 13 h, with the exception of the most lipophilic probe, [¹⁷⁷Lu]1b, with a serum half-life of less than 1 h. This probe also showed the fastest blood clearance (t_1/2 = 5.4 min), more than 2-fold faster than PEG-linked probes [¹⁷⁷Lu]3 and [¹⁷⁷Lu]4, and even 3-fold faster than the other small probes without the PEG-linker, [¹⁷⁷Lu]1a and [¹⁷⁷Lu]2. In the pretargeting experiments, tumor uptake of the lead probe [¹⁷⁷Lu]4 (~6 %ID/g) was most closely approached by [¹⁷⁷Lu]2, followed by [¹⁷⁷Lu]3 and [¹⁷⁷Lu]1a. While all the smaller and more lipophilic probes suffered from increased liver uptake, the PEG-linked probe [¹⁷⁷Lu]3 with its additional negative charge surprisingly showed the highest kidney uptake among all of the probes.

Conclusion: The in vitro performance of some of the new tetrazine probes turned out to be comparable to the established lead probe [¹⁷⁷Lu]Lu-DOTA-PEG₁₁-Tz ([¹⁷⁷Lu]4). However, tumor pretargeting studies in vivo showed lower tumor uptake and increased uptake in non-target organs.