Bioorthogonal Masking of Circulating Antibody–TCO Groups Using Tetrazine-Functionalized Dextran Polymers

Abstract

Pretargeting strategies have gained popularity for the in vivo imaging and therapy of cancer by combining antibodies with small molecule radioligands. In vivo recombination of both moieties can be achieved using the bioorthogonal inverse electron demand Diels–Alder (IEDDA) chemistry between tetrazine (Tz) and trans-cyclooctene (TCO). An issue that arises with pretargeting strategies is that while part of the antibody dose accumulates at antigen-expressing tumor tissue, there is a significant portion of the injected antibody that remains in circulation, causing a reduction in target-to-background ratios. Herein, we report the development of a novel TCO scavenger, the masking agent DP– Tz. DP–Tz is based on Tz-modified dextran polymers (DP, MW = 0.5–2 MDa). Large dextran polymers were reported to exhibit low penetration of tumor vasculature and appeared nontoxic, nonimmunogenic, and easily modifiable. Our newly developed masking agent deactivates the remaining TCO-moieties on the circulating mAbs yet does not impact the tumor uptake of the Tz-radioligand. In pretargeting studies utilizing a 68Ga-labeled tetrazine radioligand ([68Ga]Ga-NOTA-PEG11-tetrazine), DP–Tz constructs (Tz/DP ratios of 62–254) significantly increased TTB ratios from 0.8 ± 0.3 (control cohorts) to up to 5.8 ± 2.3 at 2 h postinjection. Tumor tissue delineation in PET imaging experiments employing DP–Tz is significantly increased compared to control. Uptake values of other significant organs, such as heart, lungs, pancreas, and stomach, were decreased on average by 2-fold when using DP–Tz. Overall, pretargeting experiments utilizing DP–Tz showed significantly improved tumor delineation, enhanced PET image quality, and reduced uptake in vital organs. We believe that this new masking agent is a powerful new addition to the IEDDA-based pretargeting tool box and, due to its properties, an excellent candidate for clinical translation.

Pretargeted Imaging with Gallium-68 – Improving the Binding Capability by Increasing the Number of Tetrazine Motifs

“…Intravenously injected mAb stimulates accumulation at the target interaction site but a nonnegligible amount remains in circulation increasing the background signal when administering the radioligand. In order to reduce this amount, high molecular weight TCO-scavenger molecules exhibiting low vascular permeability (clearing agents) have been established with great success improving target-to-background (TTB) ratios significantly [33,34]. The need for clearing agents was completely neglected in this study and we are fully aware that our animal model does not reflect the real situation regarding TTB ratios.…”

Harnessing 64 Cu/ 67 Cu for a theranostic approach to pretargeted radioimmunotherapy

“…Broadly speaking, this methodology consists of four steps: 1) the administration of a monoclonal antibody (mAb)-TCO immunoconjugate; 2) an interval period during which the antibody accumulates at the tumor and clears from the blood; 3) the injection of a small molecule Tz-based radioligand; and 4) the in vivo click ligation between two components, followed by the rapid clearance of any excess radioligand. This strategy has proven highly effective for pretargeted PET imaging (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49) as well as pretargeted radioimmunotherapy (PRIT) using beta-emitting ( 177 Lu) and alpha-emitting ( 225 Ac) radionuclides (50)(51)(52)(53)(54)(55). Yet as the field stands poised to make the jump from the laboratory to the clinic, it stands to reason that the development of a theranostic approach to Tz/TCO pretargeting could prove instrumental in ensuring the future success of this modality.…”

Design and preclinical evaluation of nanostars for the passive pretargeting of tumor tissue

“…With such pretargeting approaches, doses to off-target tissues are significantly decreased and high radioactive doses in the tumor can be achieved [1,3,5,6]. In addition, new bioorthogonal masking techniques -in which a masking agent is injected prior to the injection of the radioligand in order to scavenge remaining circulating antibodies -have further reduced tracer uptake in healthy tissues and improved the quality of PET images [7]. Recent advances in the development of dienophiles and tetrazines for the bioorthogonal inverse electron demand Diels-Alder (IEDDA) click reaction have accelerated the exploration of pretargeting methodologies for molecular imaging, cell tracking, drug delivery and endoradiotherapy [8].…”