TY - JOUR

T1 - Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted In Vivo Chemistry

AU - Steen, E. Johanna L.

AU - Jorgensen, Jesper T.

AU - Denk, Christoph

AU - Battisti, Umberto M.

AU - Norregaard, Kamilla

AU - Edem, Patricia E.

AU - Bratteby, Klas

AU - Shalgunov, Vladimir

AU - Wilkovitsch, Martin

AU - Svatunek, Dennis

AU - Poulie, Christian B. M.

AU - Hvass, Lars

AU - Simon, Marina

AU - Wanek, Thomas

AU - Rossin, Raffaella

AU - Robillard, Marc

AU - Kristensen, Jesper L.

AU - Mikula, Hannes

AU - Kjaer, Andreas

AU - Herth, Matthias M.

PY - 2021

Y1 - 2021

N2 - 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 logD(7.4) values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected F-18-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.

AB - 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 logD(7.4) values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected F-18-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.

KW - bioorthogonal chemistry

KW - tetrazine ligation

KW - pretargeted imaging

KW - PET

KW - fluorine-18

KW - molecular imaging

KW - TRIGGERED DRUG-RELEASE

KW - DIELS-ALDER REACTION

KW - TRANS-CYCLOOCTENE

KW - C-11-LABELED TETRAZINE

KW - IMPROVED STABILITY

KW - BIODISTRIBUTION

KW - RADIOSYNTHESIS

KW - MODULATION

KW - ANTIBODIES

U2 - 10.1021/acsptsci.1c00007

DO - 10.1021/acsptsci.1c00007

M3 - Journal article

C2 - 33860205

VL - 4

SP - 824

EP - 833

JO - ACS Pharmacology and Translational Science

JF - ACS Pharmacology and Translational Science

SN - 2575-9108

IS - 2

ER -