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

Abstract

The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo 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 in vivo 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 in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans -cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M –1 s –1 ) for the reaction with TCO and low calculated log D 7.4 values (below −3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18 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 in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

Direct Cu-mediated aromatic 18F-labeling of highly reactive tetrazines for pretargeted bioorthogonal PET imaging

“…The IEDDA reactivity is one of the most crucial factors for pretargeted in vivo applications. 14 Electrondonating and electron-withdrawing substituents were introduced on the phenyl moiety at different positions, and the substitution pattern was correlated with its synthetic accessibility and RCCs (used as a surrogate for RCYs, RCC correlated with RCY in our study) (Table 2). While all 5-substituted stannane precursors were successfully synthesized from respective iodo-Tz intermediates, only the methyl and/or methoxy derivatives among 4-and 6-substituted stannanes could be prepared -most likely due to steric hindrance.…”

“…21 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. 14,21,33 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.…”

“…[31][32][33] The latest strategy added to this portfolio is the synthesis of 18 F-radiolabeled tetrazines via the copper-catalyzed azide-alkyne cycloaddition. 14,21 Within this study, we aimed to develop a simple, scalable and reliable direct aromatic radiofluorination procedure that can be applied to access highly reactive 18 F-labeled Tzs (Figure 1). Direct aromatic [ 18 F]fluorinations are in general fast and efficient and corresponding fluoroarenes are more stable towards defluorination than their aliphatic counterparts.…”

“…[9][10][11][12][13] Improved imaging contrast (up to 100-fold) and lower radiation burden to healthy tissue can be achieved using pretargeting compared to conventional imaging strategies. 14 These improved imaging characteristics are a result of the temporal separation of the slow targeting process of nanomedicines from the actual imaging step. Consequently, the exceptional target specificity of nanomedicines as well as the optimal pharmacokinetics of small molecules for molecular imaging, e.g.…”

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications

“…The di-aryl substituted tetrazine, 3,6-di-(2-pyridyl)-1,2,4,5-tetrazine 10 , is the diene that exhibited the worst stability, as well as the best reactivity among all tetrazines tested. Recently, Steen et al developed a library of 45 tetrazines and compared their lipophilicity (clogD 7.4 ), topological polar surface areas (TPSAs) and in vivo IEDDA reactivity [ 74 ]. A strong correlation was noticed between the tetrazine ligation efficiency and the lipophilicity of the Tz.…”