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.