Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted in Vivo Chemistry

Abstract

<p>The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of <i>in vivo</i> chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile <i>in vivo</i> chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the <i>in vivo</i> ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the <i>in vivo</i> fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted <i>trans</i>-cyclooctene (TCO)-tagged antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their <i>in vivo</i> performance. In particular, high rate constants (>50,000 M<sup>-1</sup>s<sup>-1</sup>) for the reaction with TCO and low calculated log<i>D</i><sub>7.4</sub> values (below -3) of the tetrazine were identified as strong indicators for successful pretargeted <i>in vivo</i> click chemistry. Click-radiolabeling gave access to a set of selected <sup>18</sup>F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for <i>in vivo</i> application and will thereby assist the clinical translation of bioorthogonal pretargeting.</p>

Development of the First Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging – Expanding the Bioorthogonal Tool Box

“…[19][20][21][22] From a clinical point of view, fluorine-18 is an almost ideal radionuclide for Positron-Emission-Tomography (PET). [23][24][25] Its unique decay characteristics give rise to high resolution PET images and result in acceptable levels of radiation burden. In addition, the half-life of 110 min also enables distribution from the production site to other research facilities and clinics, and allows, as such, for commercialization.…”

“…In addition, the half-life of 110 min also enables distribution from the production site to other research facilities and clinics, and allows, as such, for commercialization. [23][24][25] We have recently reported the first successful direct labeling of 18 F-Tzs with suitable reactivity for in vivo chemistry. 26 In another study, we identified key properties for Tzs to be used for pretargeted strategies.…”

“…26 In another study, we identified key properties for Tzs to be used for pretargeted strategies. 25 In this study, we combined this knowledge and developed the first direct aliphatic 18 F-radiolabeled Tz suitable for pretargeted PET imaging. This tracer could potentially be used for dose estimations before pretargeted radionuclide approaches are initiated.…”

Direct Aromatic 18F-Labeling of Highly Reactive Tetrazines for Pretargeted Bioorthogonal PET Imaging

“…[17] The reason for this is that highly reactive mono-or bis-(hetero)aryl-substituted Tzs decompose under the harsh conditions used for standard nucleophilic 18 F-fluorination (SN2 or SNAr) approaches. [11,17,29] Only relatively base insensitive and less reactive Tzs could be radiolabeled, via an 18 F-aliphatic substitution (SN2) strategy. Radiochemical yields (RCYs) up to 18% were achieved.…”

“…The mean tumor-to-blood ratio was 0.9, and thereby the specific uptake is similar to what was previously reported for other pretargeted imaging agents in the same tumor model. [11] In contrast, a mean tumor-to-muscle ratio of 10 was detected which in fact is significantly higher compared to what has previously been found for the "state-of-the-art" Tz-based imaging agents [ 18 F]22 and [ 64 Cu]Cu-NOTA-PEG7-H-Tzin a similar pretargeting set-up (LS174T bearing mice, using CC49-TCO 72h prior to tracer injection, similar imaging timeframes) (Figure 4C and D). [25,59] However, [ 18 F]21 showed a 3 to 5-fold lower tumor uptake compared to those imaging agents (Figure 4E).…”