Mice and men: An antibody conjugate with trans‐cyclooctene was administered to tumor‐bearing mice, and the resulting chemically tagged tumors were subsequently treated with an 111In‐labeled tetrazine ...probe in an inverse‐electron‐demand Diels–Alder reaction. The adduct was formed in a remarkable 52–57 % yield in vivo and used for non‐invasive pretargeted tumor imaging in mice (see picture).
•Non-invasive molecular imaging and therapeutic applications in living mice.•System requirements.•Comparison of several click reactions.•Future challenges.
Following the successful application of in ...vivo chemistry in chemical biology there has been growing interest in extending the application scope to non-invasive molecular imaging and therapy in living animals and eventually humans. A typical example of such an application is pretargeted radioimmuno-imaging and -therapy: the tumor targeting of a tagged antibody followed by administration and binding of a small radiolabeled probe to the tag of the tumor-bound antibody. In this review, we describe the requirements to take the step to non-invasive applications in animals, summarize recent achievements in this field, and we offer a perspective on future developments.
Eliminated without a trace: The fastest click reaction, the highly selective inverse‐electron‐demand Diels–Alder reaction, has been modified to enable selective bioorthogonal release. Thus, the click ...reaction of a tetrazine with a drug‐bound trans‐cyclooctene caused the instantaneous release of the drug and CO2 (see scheme). One possible application is the chemically triggered release, and thereby activation, of a drug from a tumor‐bound antibody–drug conjugate.
Current antibody-drug conjugates (ADCs) target internalising receptors on cancer cells leading to intracellular drug release. Typically, only a subset of patients with solid tumours has sufficient ...expression of such a receptor, while there are suitable non-internalising receptors and stroma targets. Here, we demonstrate potent therapy in murine tumour models using a non-internalising ADC that releases its drugs upon a click reaction with a chemical activator, which is administered in a second step. This was enabled by the development of a diabody-based ADC with a high tumour uptake and very low retention in healthy tissues, allowing systemic administration of the activator 2 days later, leading to efficient and selective activation throughout the tumour. In contrast, the analogous ADC comprising the protease-cleavable linker used in the FDA approved ADC Adcetris is not effective in these tumour models. This first-in-class ADC holds promise for a broader applicability of ADCs across patient populations.
The bioorthogonal cleavage of allylic carbamates from trans‐cyclooctene (TCO) upon reaction with tetrazine is widely used to release amines. We disclose herein that this reaction can also cleave TCO ...esters, carbonates, and surprisingly, ethers. Mechanistic studies demonstrated that the elimination is mainly governed by the formation of the rapidly eliminating 1,4‐dihydropyridazine tautomer, and less by the nature of the leaving group. In contrast to the widely used p‐aminobenzyloxy linker, which affords cleavage of aromatic but not of aliphatic ethers, the aromatic, benzylic, and aliphatic TCO ethers were cleaved as efficiently as the carbamate, carbonate, and esters. Bioorthogonal ether release was demonstrated by the rapid uncaging of TCO‐masked tyrosine in serum, followed by oxidation by tyrosinase. Finally, tyrosine uncaging was used to chemically control cell growth in tyrosine‐free medium.
Click‐to‐release… more: In addition to cleaving carbamates, the inverse‐electron‐demand Diels–Alder pyridazine elimination reaction can, with the same efficiency, cleave aromatic, benzylic, and even aliphatic ethers, as well as esters and carbonates. trans‐Cyclooctene‐caged tyrosine was rapidly uncaged by treatment with tetrazine and subsequently oxidized by tyrosinase, whereas the caged tyrosine itself was not a substrate.
The high rate of the ‘click-to-release’ reaction between an allylic substituted trans-cyclooctene linker and a tetrazine activator has enabled exceptional control over chemical and biological ...processes. Here we report the development of a new bioorthogonal cleavage reaction based on trans-cyclooctene and tetrazine, which allows the use of highly reactive trans-cyclooctenes, leading to 3 orders of magnitude higher click rates compared to the parent reaction, and 4 to 6 orders higher than other cleavage reactions. In this new pyridazine elimination mechanism, wherein the roles are reversed, a trans-cyclooctene activator reacts with a tetrazine linker that is substituted with a methylene-linked carbamate, leading to a 1,4-elimination of the carbamate and liberation of a secondary amine. Through a series of mechanistic studies, we identified the 2,5-dihydropyridazine tautomer as the releasing species and found factors that govern its formation and subsequent fragmentation. The bioorthogonal utility was demonstrated by the selective cleavage of a tetrazine-linked antibody–drug conjugate by trans-cyclooctenes, affording efficient drug liberation in plasma and cell culture. Finally, the parent and the new reaction were compared at low concentration, showing that the use of a highly reactive trans-cyclooctene as the activator leads to a complete cycloaddition reaction with the antibody–drug conjugate in seconds vs hours for the parent system. Although the subsequent release from the IEDDA adduct is slower, we believe that this new reaction may allow markedly reduced click-to-release reagent doses in vitro and in vivo and could expand the application scope to conditions wherein the trans-cyclooctene has limited stability.
Current pretargeting systems use noncovalent biologic interactions, which are prone to immunogenicity. We previously developed a novel approach based on the bioorthogonal reaction between a ...radiolabeled tetrazine and an antibody-conjugated trans-cyclooctene (TCO). However, the tumor-to-blood ratio was low due to reaction with freely circulating antibody-TCO.
Here we developed 2 tetrazine-functionalized clearing agents that enable rapid reaction with and removal of a TCO-tagged antibody (CC49) from blood. Next, we incorporated this approach into an optimized pretargeting protocol in LS174T-bearing mice. Then we compared the pretargeted (177)Lu-labeled tetrazine with (177)Lu-labeled CC49. The biodistribution data were used for mouse and human dosimetry calculations.
The use of a clearing agent led to a doubling of the tetrazine tumor uptake and a 125-fold improvement of the tumor-to-blood ratio at 3 h after tetrazine injection. Mouse dosimetry suggested that this should allow for an 8-fold higher tumor dose than is possible with nonpretargeted radioimmunotherapy. Also, humans treated with CC49-TCO-pretargeted (177)Lu-tetrazine would receive a dose to nontarget tissues 1 to 2 orders of magnitude lower than with directly labeled CC49.
The in vivo performance of chemical pretargeting falls within the range of results obtained for the clinically validated pretargeting approaches in mice, with the advantage of potentially allowing for fractionated radiotherapy as a result of a lower likelihood of immunogenicity. These findings demonstrate that biologic pretargeting concepts can be translated to rapid bioorthogonal chemical approaches with retained potential.
One of the challenges of pretargeted radioimmunotherapy, which centers on the capture of a radiolabeled probe by a preinjected tumor-bound antibody, is the potential immunogenicity of biological ...capturing systems. A bioorthogonal chemical approach may circumvent this drawback, but effective in vivo chemistry in mice, larger animals, and eventually humans, requires very high reagent reactivity, sufficient stability, and retained selectivity. We report here that the reactivity of the fastest bioorthogonal reaction, the inverse-electron-demand-Diels–Alder cycloaddition between a tetrazine probe and a trans-cyclooctene-tagged antibody, can be increased 10-fold (k 2 = 2.7 × 105 M–1 s–1) via the trans-cyclooctene, approaching the speed of biological interactions, while also increasing its stability. This was enabled by the finding that the trans-cyclooctene tag is probably deactivated through isomerization to the unreactive cis-cyclooctene isomer by interactions with copper-containing proteins, and that increasing the steric hindrance on the tag can impede this process. Next, we found that the higher reactivity of axial vs equatorial linked TCO can be augmented by the choice of linker. The new, stabilized, and more reactive tag allowed for improved tumor-to-nontumor ratios in pretargeted tumor-bearing mice.
Tumor targeting using agents with slow pharmacokinetics represents a major challenge in nuclear imaging and targeted radionuclide therapy as they most often result in low imaging contrast and high ...radiation dose to healthy tissue. To address this challenge, we developed a polymer-based targeting agent that can be used for pretargeted imaging and thus separates tumor accumulation from the imaging step in time. The developed targeting agent is based on polypeptide-graft-polypeptoid polymers (PeptoBrushes) functionalized with trans-cyclooctene (TCO). The complementary 111In-labeled imaging agent is a 1,2,4,5-tetrazine derivative, which can react with aforementioned TCO-modified PeptoBrushes in a rapid bioorthogonal ligation. A high degree of TCO loading (up to 30%) was achieved, without altering the physicochemical properties of the polymeric nanoparticle. The highest degree of TCO loading resulted in significantly increased reaction rates (77-fold enhancement) compared to those with small molecule TCO moieties when using lipophilic tetrazines. Based on computer simulations, we hypothesize that this increase is a result of hydrophobic effects and significant rearrangements within the polymer framework, in which hydrophobic patches of TCO moieties are formed. These patches attract lipophilic tetrazines, leading to increased reaction rates in the bioorthogonal ligation. The most reactive system was evaluated as a targeting agent for pretargeted imaging in tumor-bearing mice. After the setup was optimized, sufficient tumor-to-background ratios were achieved as early as 2 h after administration of the tetrazine imaging agent, which further improved at 22 h, enabling clear visualization of CT-26 tumors. These findings show the potential of PeptoBrushes to be used as a pretargeting agent when an optimized dose of polymer is used.
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