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).

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.

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 (k2 = 2.7 × 10(5) 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.

The use of a bioorthogonal reaction for the selective cleavage of tumor-bound antibody-drug conjugates (ADCs) would represent a powerful new tool for ADC therapy, as it would not rely on the currently used intracellular biological activation mechanisms, thereby expanding the scope to noninternalizing cancer targets. Here we report that the recently developed inverse-electron-demand Diels-Alder pyridazine elimination reaction can provoke rapid and self-immolative release of doxorubicin from an ADC in vitro and in tumor-bearing mice.

A biodegradable positron-emitting dendritic nanoprobe targeted at ␣v␤3 integrin, a biological marker known to modulate angiogenesis, was developed for the noninvasive imaging of angiogenesis. The nanoprobe has a modular multivalent core-shell architecture consisting of a biodegradable heterobifunctional dendritic core chemoselectively functionalized with heterobifunctional polyethylene oxide (PEO) chains that form a protective shell, which imparts biological stealth and dictates the pharmacokinetics. Each of the 8 branches of the dendritic core was functionalized for labeling with radiohalogens. Placement of radioactive moieties at the core was designed to prevent in vivo dehalogenation, a potential problem for radiohalogens in imaging and therapy. Targeting peptides of cyclic arginine-glycine-aspartic acid (RGD) motifs were installed at the terminal ends of the PEO chains to enhance their accessibility to ␣v␤3 integrin receptors. This nanoscale design enabled a 50-fold enhancement of the binding affinity to ␣v␤3 integrin receptors with respect to the monovalent RGD peptide alone, from 10.40 nM to 0.18 nM IC50. Cell-based assays of the 125 I-labeled dendritic nanoprobes using ␣v␤3-positive cells showed a 6-fold increase in ␣v␤3 receptor-mediated endocytosis of the targeted nanoprobe compared with the nontargeted nanoprobe, whereas ␣v␤3-negative cells showed no enhancement of cell uptake over time. In vivo biodistribution studies of 76 Brlabeled dendritic nanoprobes showed excellent bioavailability for the targeted and nontargeted nanoprobes. In vivo studies in a murine hindlimb ischemia model for angiogenesis revealed high specific accumulation of 76 Br-labeled dendritic nanoprobes targeted at ␣v␤3 integrins in angiogenic muscles, allowing highly selective imaging of this critically important process.dendrimer ͉ molecular imaging

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. Methods: 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. Results: 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. Conclusion: 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.

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.

Wie sieht's aus in der Maus? Ein trans‐Cycloocten‐Antikörper‐Konjugat wurde Tumormäusen verabreicht. Die derart chemisch markierten Tumore wurden dann mit einer 111In‐markierten Tetrazin‐Sonde in einer Diels‐Alder‐Reaktion mit inversem Elektronenbedarf umgesetzt, die das Addukt in vivo mit einer bemerkenswerten Ausbeute von 52–57 % ergab. Mit diesem nichtinvasiven Verfahren gelang die Abbildung von Tumoren in Mäusen (siehe Bild).