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.

Pretargeted imaging of nanomedicines have attracted considerable interest because it has the potential to increase imaging contrast while reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction for this strategy and, consequently, the state-of-art choice for in vivo chemistry. We have recently identified key properties for tetrazines in pretargeting. We have also developed a method to 18 F-label reactive tetrazines using an aliphatic nucleophilic substitution strategy. Here, we combined this knowledge and developed an 18 F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small SAR study was performed. The most promising compound was selected for labeling and subsequent positronemission-tomography in vivo imaging. Radiolabeling was achieved in satisfactory yields, molar activities, and high radiochemical purities. [ 18 F]15 displayed favorable pharmacokinetics and remarkable target-to-background ratiosas early as 1 h post injection. We believe that this agent could be a promising candidate for translation into clinical studies.

Aliphatic nucleophilic substitution (S N 2) with [ 18 F]fluoride is the most widely applied method to prepare 18 F-labeled positron emission tomography (PET) tracers. Strong basic conditions commonly used during 18 F-labeling procedures inherently limit or prohibit labeling of base-sensitive scaffolds. The high basicity stems from the tradition to trap [ 18 F]fluoride on anion exchange cartridges and elute it afterward with basic anions. This sequence is used to facilitate the transfer of [ 18 F]fluoride from an aqueous to an aprotic organic, polar reaction medium, which is beneficial for S N 2 reactions. Furthermore, this sequence also removes cationic radioactive contaminations from cyclotron-irradiated [ 18 O]water from which [ 18 F]fluoride is produced. In this study, we developed an efficient elution procedure resulting in low basicity that permits S N 2 18 F-labeling of base-sensitive scaffolds. Extensive screening of trapping and elution conditions (>1000 experiments) and studying their influence on the radiochemical yield (RCY) allowed us to identify a suitable procedure for this. Using this procedure, four PET tracers and three synthons could be radiolabeled in substantially higher RCYs (up to 2.5-fold) compared to those of previously published procedures, even from lower precursor amounts. Encouraged by these results, we applied our low-basicity method to the radiolabeling of highly base-sensitive tetrazines, which cannot be labeled using state-of-art direct aliphatic 18 F-labeling procedures. Labeling succeeded in RCYs of up to 20%. We believe that our findings facilitate PET tracer development by opening the path toward simple and direct S N 2 18 F fluorination of base-sensitive substrates.

<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^-1s^-1) for the reaction with TCO and low calculated log<i>D</i>_7.4 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 ¹⁸F-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>

Pretargeted PET imaging is an emerging and fast-developing method to monitor immuno-oncology strategies. Currently, tetrazine ligation is considered the most promising bioorthogonal reaction for pretargeting in vivo. Recently, we have developed a method to 18F-label ultrareactive tetrazines by copper-mediated fluorinations. However, bispyridyl tetrazines—one of the most promising structures for in vivo pretargeted applications—were inaccessible using this strategy. We believed that our successful efforts to 18F-label H-tetrazines using low basic labeling conditions could also be used to label bispyridyl tetrazines via aliphatic nucleophilic substitution. Here, we report the first direct 18F-labeling of bispyridyl tetrazines, their optimization for in vivo use, as well as their successful application in pretargeted PET imaging. This strategy resulted in the design of [18F]45, which could be labeled in a satisfactorily radiochemical yield (RCY = 16%), molar activity (Am = 57 GBq/µmol), and high radiochemical purity (RCP > 98%). The [18F]45 displayed a target-to-background ratio comparable to previously successfully applied tracers for pretargeted imaging. This study showed that bispyridyl tetrazines can be developed into pretargeted imaging agents. These structures allow an easy chemical modification of 18F-labeled tetrazines, paving the road toward highly functionalized pretargeting tools. Moreover, bispyridyl tetrazines led to near-instant drug release of iTCO-tetrazine-based ‘click-to-release’ reactions. Consequently, 18F-labeled bispyridyl tetrazines bear the possibility to quantify such release in vivo in the future.

Glycomimetic drugs have attracted increasing interest as unique targeting vectors or surrogates for endogenous biomolecules. However, it is generally difficult to determine the in vivo pharmacokinetic profile of these compounds. In this work, two galectin-3 inhibitors were radiolabeled with fluorine-18 and used as surrogate PET tracers of TD139 and GB1107. Both compounds are promising drugs for clinical applications. In vivo evaluation revealed that both surrogates strongly differed with respect to their biodistribution profile. The disaccharide (TD139 surrogate) was rapidly eliminated from blood while the monosaccharide (GB1107 surrogate) showed no sign of excretion. The data obtained allowed us to infer the different in vivo fate of TD139 and GB1107 and rationalize how different administration routes could boost efficacy. Whereas the fast excretion profile of the TD139 surrogate indicated that systemic application of disaccharides is unfavorable, the extended biological half-life of the GB1107 surrogate indicated that systemic administration is possible for monosaccharides.

BackgroundTo study the ability of the newly developed α‐synuclein Positron Emission Tomography (PET) tracer, [18F]ACI‐12589, created and developed by AC Immune SA, to detect α‐synuclein neuropathology in vivo, in clinically diagnosed patients with α‐synucleinopathies.MethodWe intend to scan a total of up to 50 participants with different diseases associated with α‐synuclein and age‐ and gender‐matched controls. These will include participants with Parkinson’s disease (PD), Multiple System Atrophy (MSA), Dementia with Lewy bodies (DLB), or Parkinson’s disease caused by a duplication in the α‐synuclein gene, in addition to neurologically healthy volunteers. At the time of abstract submission, 23 participants have undergone [18F]ACI‐12589 PET scans. 20 scans consist of 0‐90 min dynamic scans with arterial blood sampling for blood input activity and metabolite analysis, two are full dynamic scans and one a 60‐90 min static scan without blood sampling. In addition, participants have undergone structural MRI, DaTscan or [18F]DOPA PET, and cognitive and motor testing (UPDRS‐III).ResultThe data are currently being processed and analyzed. Initial image analyses suggest that there is ACI‐12589 retention in brain areas affected by the disease process (such as the basal ganglia and cerebellar white matter) in participants with MSA, but also a potential age‐dependent retention in the basal ganglia and brain stem of controls.ConclusionWe will be presenting initial promising clinical data on the novel α‐synuclein PET tracer [18F]ACI‐12589, the initial analysis of which suggests tracer binding consistent with expected patterns of α‐synuclein pathology based on clinical manifestations. Further analyses are ongoing and our updated findings will be presented at the conference.

Aliphatic nucleophilic substitution (S_N2) with [¹⁸F]fluoride is the most widely applied method to prepare ¹⁸F-labeled positron emission tomography (PET) tracers. Strongly basic conditions commonly used during ¹⁸F-labeling procedures inherently limit or prohibit labeling of base-sensitive scaffolds. The high basicity stems from the tradition to trap [¹⁸F]fluoride on anion exchange cartridges and elute it afterwards with basic anions. This sequence is used to facilitate the transfer of [¹⁸F]fluoride from an aqueous to an aprotic organic, polar reaction medium, which is beneficial for S_N2 reactions. Furthermore, this sequence also removes cationic radioactive contaminations from cyclotron-irradiated [¹⁸O]water from which [¹⁸F]fluoride is produced. In this study, we developed an efficient elution procedure resulting in low basicity that permits S_N2 ¹⁸F-labeling of base-sensitive scaffolds. Extensive screening of trapping and elution conditions (>1000 experiments) and studying their influence on the radiochemical yield (RCY) allowed us to identify a suitable procedure for this. Four PET tracers and three synthons could be radiolabeled in substantially higher RCYs (up to 2.5-fold), even from lower precursor amounts, using this procedure. Encouraged by these results, we applied our low basicity method to the radiolabeling of highly base-sensitive tetrazines, which cannot be labeled using state-of-art direct aliphatic ¹⁸F-labeling procedures. Labeling succeeded in RCYs of up to 20%. We believe that our findings facilitate PET tracer development by opening the path towards simple and direct S_N2 ¹⁸F-fluorination of base-sensitive substrates.