Reactive polymer enables efficient in vivo bioorthogonal chemistry

Abstract

There has been intense interest in the development of selective bioorthogonal reactions or "click" chemistry that can proceed in live animals. Until now however, most reactions still require vast surpluses of reactants because of steep temporal and spatial concentration gradients. Using computational modeling and design of pharmacokinetically optimized reactants, we have developed a predictable method for efficient in vivo click reactions. Specifically, we show that polymer modified tetrazines (PMT) are a key enabler for in vivo bioorthogonal chemistry based on the very fast and catalyst-free [4 þ 2] tetrazine/trans-cyclooctene cycloaddition. Using fluorescent PMT for cellular resolution and 18 F labeled PMT for whole animal imaging, we show that cancer cell epitopes can be easily reacted in vivo. This generic strategy should help guide the design of future chemistries and find widespread use for different in vivo bioorthogonal applications, particularly in the biomedical sciences.in vivo chemistry | pharmacokinetics | PET imaging | intravital microscopy | pretargeting T he ability to perform selective chemistries in living systems such as single cells, 3D cultures, invertebrates, or mammals would have far reaching applications in tracking biomolecules, designing new therapeutic approaches, and in visualizing medically relevant biomarkers. To date, only a few practical bioorthogonal reactions have been reported, the most popular being the Staudinger ligation and the [3 þ 2] cycloaddition "click" reaction between azides and alkynes (1, 2). The latter click reaction involves copper(I) catalyzed coupling of an azide and terminal alkyne to generate a stable triazole (2). Until recently, the necessity of the copper catalyst precluded use of this reaction in biological systems due to toxicity concerns (3, 4). Bertozzi and others elegantly solved this problem by developing several new ring strained dienophile derivatives that do not require catalysts (5-9). However, many of these derivatives have low water solubility, require complex multistep synthesis, and possess suboptimal kinetics. Our search for alternative rapid, selective, and chemically accessible coupling reactions without need for a catalyst led us and others to investigate the [4 þ 2] inverse Diels-Alder cycloaddition (10-13). We realized that this set of chemistries is more uniquely suited to biological applications and may indeed represent a universal platform technology ( Fig. 1 A and B). Specifically we and others have shown that the cycloaddition between tetrazine (Tz) and trans-cyclooctene (TCO) can proceed orders of magnitude faster than previously studied azide and alkyne click reactions and importantly does not require the action of a catalyst. This reaction has also been adapted for single cell imaging using newer fluorogenic Tz probes (14).Although initial work focused on in vitro labeling there has been a surge of recent work applying this reaction for various in vivo applications (15-17). Despite the above progress, our results with initial TCO...

18F-Labeling of Mannan for Inflammation Research with Positron Emission Tomography

“…16 In another work, tetrazine-bearing dextrans have been conjugated to 18 F-trans-cyclooctene ([ 18 F]TCO, Figure 1) based on inverse electron demand Diels−Alder (IEDDA) reaction. 17 IEDDA reaction has emerged as the fastest chemistry scheme, and tetrazine-trans-cyclooctene ligation has been widely applied in different technologies. 18 Tetrazine-trans-cyclooctene ligation may generate products with multiple isomers.…”

Diels–Alder Reaction for Tumor Pretargeting: In Vivo Chemistry Can Boost Tumor Radiation Dose Compared with Directly Labeled Antibody

“…Such a chemical approach is less likely to give rise to immunogenicity and may in addition enable universal and straightforward tagging and in vivo tracking of mAbs without severe perturbation of the in vivo properties of the parent construct. Although the use of organic chemistry in living beings is a highly sought-after approach, only the inverse-electron-demand Diels-Alder reaction between strained trans-cyclooctene (TCO) and electron-deficient tetrazines has so far demonstrated sufficient potential for such demanding conditions (6)(7)(8)(9)(10). We reported previously that the use of this reaction could indeed be extended to a living system, showing pronounced and specific localization of an 111 In-labeled DOTAtetrazine probe in LS174T-tumored mice that were pretargeted with anti-TAG72 mAb CC49-TCO (8).…”

Bioorthogonal chemistries for nanomaterial conjugation and targeting

“…Another approach, however, would be to employ multiple bioorthogonal chemistries that are orthogonal to each other in concert. One such mutually orthogonal pairing has already been demonstrated for TCO/Tz and cyclooctyne/azide chemistries [30] . Finally, Deveraj et al have established that the TCO/Tz chemistry can be utilized for in vivo detection of cancer using separate injections of TCO-antibody and a Tz-modified fluorescent polymer.…”

“…reaction between Tz and TCO has enabled live cell labeling both in vitro [28] and in vivo [29,30] . Interestingly, the TCO/Tz chemistry has been shown to be orthogonal to octyne-azide if a slower-reacting Tz is employed [31] .…”