Click-to-Release: Cleavable Radioimmunoimaging with [89Zr]Zr-DFO-Trans-Cyclooctene-Trastuzumab Increases Tumor-to-Blood Ratio.

One of the main challenges of PET imaging with 89Zr-labeled monoclonal antibodies (mAbs) remains the long blood circulation of the radiolabeled mAbs, leading to high background signals, decreasing image quality. To overcome this limitation, here we report the use of a bioorthogonal linker cleavage approach (click-to-release chemistry) to selectively liberate [89Zr]Zr-DFO from trans-cyclooctene-functionalized trastuzumab (TCO-Tmab) in blood, following the administration of a tetrazine compound (trigger) in BT-474 tumor-bearing mice. Methods: We created a series of TCO-DFO constructs and evaluated their performance in [89Zr]Zr-DFO release from Tmab in vitro using different trigger compounds. The in vivo behavior of the best performing [89Zr]Zr-TCO-Tmab was studied in healthy mice first to determine the optimal dose of the trigger. To find the optimal time for the trigger administration, the rate of [89Zr]Zr-TCO-Tmab internalization was studied in BT-474 cancer cells. Finally, the trigger was administered 6 h or 24 h after [89Zr]Zr-TCO-Tmab- administration in tumor-bearing mice to liberate the [89Zr]Zr-DFO fragment. PET scans were obtained of tumor-bearing mice that received the trigger 6 h post-[89Zr]Zr-TCO-Tmab administration. Results: The [89Zr]Zr-TCO-Tmab and trigger pair with the best in vivo properties exhibited 83% release in 50% mouse plasma. In tumor-bearing mice the tumor-blood ratios were markedly increased from 1.0 ± 0.4 to 2.3 ± 0.6 (p = 0.0057) and from 2.5 ± 0.7 to 6.6 ± 0.9 (p < 0.0001) when the trigger was administered at 6 h and 24 h post-mAb, respectively. Same day PET imaging clearly showed uptake in the tumor combined with a strongly reduced background due to the fast clearance of the released [89Zr]Zr-DFO-containing fragment from the circulation through the kidneys. Conclusions: This is the first demonstration of the use of trans-cyclooctene-tetrazine click-to-release chemistry to release a radioactive chelator from a mAb in mice to increase tumor-to-blood ratios. Our results suggest that click-cleavable radioimmunoimaging may allow for substantially shorter intervals in PET imaging with full mAbs, reducing radiation doses and potentially even enabling same day imaging.

A Concise Synthetic Approach to Highly Reactive Click-to-Release Trans-Cyclooctene Linkers.

An increase in the click-to-release reaction rate between cleavable trans-cyclooctenes (TCO) and tetrazines would be beneficial for drug delivery applications. In this work, we have developed a short and stereoselective synthesis route towards highly reactive sTCOs that serve as cleavable linkers, affording quantitative tetrazine-triggered payload release. In addition, the fivefold more reactive sTCO exhibited the same in vivo stability as current TCO linkers when used as antibody linkers in circulation in mice.

Functionalization of Radiolabeled Antibodies to Enhance Peripheral Clearance for High Contrast Brain Imaging.

Small molecule imaging agents such as [11C]PiB, which bind to the core of insoluble amyloid-ß (Aß) fibrils, are useful tools in Alzheimer's disease (AD) research, diagnostics, and drug development. However, the [11C]PiB PET signal saturates early in the disease progression and does not detect soluble or diffuse Aß pathology which are believed to play important roles in the disease progression. Antibodies, modified into a bispecific format to enter the brain via receptor-mediated transcytosis, could be a suitable alternative because of their diversity and high specificity for their target. However, the circulation time of these antibodies is long, resulting in an extended exposure to radiation and low imaging contrast. Here, we explore two alternative strategies to enhance imaging contrast by increasing clearance of the antibody ligand from blood. The bispecific Aß targeting antibody RmAb158-scFv8D3 and the monospecific RmAb158 were radiolabeled and functionalized with either α-d-mannopyranosylphenyl isothiocyanate (mannose) or with trans-cyclooctene (TCO). While mannose can directly mediate antibody clearance via the liver, TCO-modified antibody clearance was induced by injection of a tetrazine-functionalized, liver-targeting clearing agent (CA). In vivo experiments in wild type and AD transgenic mice demonstrated the ability of both strategies to drastically shorten the circulation time of RmAb158, while they had limited effect on the bispecific variant RmAb158-8D3. Furthermore, single photon emission computed tomography imaging with TCO-[125I]I-RmAb158 in AD mice showed higher contrast 1 day after injection of the tetrazine-functionalized CA. In conclusion, strategies to enhance the clearance of antibody-based imaging ligands could allow imaging at earlier time points and thereby open the possibility to combine antibodies with short-lived radionuclides such as fluorine-18.

Bioorthogonal chemistry.

Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups. Rooted in the principles of physical organic chemistry, bioorthogonal reactions are intrinsically selective transformations not commonly found in biology. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide-alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions. Bioorthogonal chemistry has significant overlap with the broader field of 'click chemistry' - high-yielding reactions that are wide in scope and simple to perform, as recently exemplified by sulfuryl fluoride exchange chemistry. The underlying mechanisms of these transformations and their optimal conditions are described in this Primer, followed by discussion of how bioorthogonal chemistry has become essential to the fields of biomedical imaging, medicinal chemistry, protein synthesis, polymer science, materials science and surface science. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody-drug conjugation and drug delivery. This Primer describes standards for reproducibility and data deposition, outlines how current limitations are driving new research directions and discusses new opportunities for applying bioorthogonal chemistry to emerging problems in biology and biomedicine.

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

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 18F-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.

Bioorthogonal Tetrazine Carbamate Cleavage by Highly Reactive trans-Cyclooctene.

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.

Fluorogenic Bifunctional trans-Cyclooctenes as Efficient Tools for Investigating Click-to-Release Kinetics.

The inverse electron demand Diels-Alder pyridazine elimination reaction between tetrazines and allylic substituted trans-cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination of alkylamine substrates has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO-linked EDANS fluorophore and a DABCYL quencher for accurate determination of both the click and release rate constants for any tetrazine at physiologically relevant concentrations.

Evaluation of a 68Ga-Labeled DOTA-Tetrazine as a PET Alternative to 111In-SPECT Pretargeted Imaging.

The bioorthogonal reaction between a tetrazine and strained transcyclooctene (TCO) has garnered success in pretargeted imaging. This reaction was first validated in nuclear imaging using an 111In-labeled 1,4,7,10tetraazacyclododecane1,4,7,10tetraacetic acid (DOTA)-linked bispyridyl tetrazine (Tz) ([111In]In-DOTA-PEG11-Tz) and a TCO functionalized CC49 antibody. Given the initial success of this Tz, it has been paired with TCO functionalized small molecules, diabodies, and affibodies for in vivo pretargeted studies. Furthermore, the single photon emission tomography (SPECT) radionuclide, 111In, has been replaced with the ß-emitter, 177Lu and α-emitter, 212Pb, both yielding the opportunity for targeted radiotherapy. Despite use of the 'universal chelator', DOTA, there is yet to be an analogue suitable for positron emission tomography (PET) using a widely available radionuclide. Here, a 68Ga-labeled variant ([68Ga]Ga-DOTA-PEG11-Tz) was developed and evaluated using two different in vivo pretargeting systems (Aln-TCO and TCO-CC49). Small animal imaging and ex vivo biodistribution studies were performed and revealed target specific uptake of [68Ga]Ga-DOTA-PEG11-Tz in the bone (3.7 %ID/g, knee) in mice pretreated with Aln-TCO and tumor specific uptake (5.8 %ID/g) with TCO-CC49 in mice bearing LS174 xenografts. Given the results of this study, [68Ga]Ga-DOTA-PEG11-Tz can serve as an alternative to [111In]In-DOTA-PEG11-Tz.

Trans-Cyclooctene-Functionalized PeptoBrushes with Improved Reaction Kinetics of the Tetrazine Ligation for Pretargeted Nuclear Imaging.

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.

Evaluation of the inverse electron demand Diels-Alder reaction in rats using a scandium-44-labelled tetrazine for pretargeted PET imaging.

BACKGROUND: Pretargeted imaging allows the use of short-lived radionuclides when imaging the accumulation of slow clearing targeting agents such as antibodies. The biotin-(strept)avidin and the bispecific antibody-hapten interactions have been applied in clinical pretargeting studies; unfortunately, these systems led to immunogenic responses in patients. The inverse electron demand Diels-Alder (IEDDA) reaction between a radiolabelled tetrazine (Tz) and a trans-cyclooctene (TCO)-functionalized targeting vector is a promising alternative for clinical pretargeted imaging due to its fast reaction kinetics. This strategy was first applied in nuclear medicine using an 111In-labelled Tz to image TCO-functionalized antibodies in tumour-bearing mice. Since then, the IEDDA has been used extensively in pretargeted nuclear imaging and radiotherapy; however, these studies have only been performed in mice. Herein, we report the 44Sc labelling of a Tz and evaluate it in pretargeted imaging in Wistar rats. RESULTS: 44Sc was obtained from an in house 44Ti/44Sc generator. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized tetrazine was radiolabelled with 44Sc resulting in radiochemical yields of 85-95%, a radiochemical purity > 99% at an apparent molar activity of 1 GBq/mmol. The 44Sc-labelled Tz maintained stability in solution for up to 24 h. A TCO-functionalized bisphosphonate, which accumulates in skeletal tissue, was used as a targeting vector to evaluate the 44Sc-labelled Tz. Biodistribution data of the 44Sc-labelled Tz showed specific uptake (0.9 ± 0.3% ID/g) in the bones (humerus and femur) of rats pre-treated with the TCO-functionalized bisphosphonate. This uptake was not present in rats not receiving pre-treatment (< 0.03% ID/g). CONCLUSIONS: We have prepared a 44Sc-labelled Tz and used it in pretargeted PET imaging with rats treated with TCO-functionalized bisphosponates. This allowed for the evaluation of the IEDDA reaction in animals larger than a typical mouse. Non-target accumulation was low, and there was a 30-fold higher bone uptake in the pre-treated rats compared to the non-treated controls. Given its convenient half-life and the ability to perform positron emission tomography with a previously studied DOTA-functionalized Tz, scandium-44 (t1/2 = 3.97 h) proved to be a suitable radioisotope for this study.

Tetrazine- trans-Cyclooctene Chemistry Applied to Fabricate Self-Assembled Fluorescent and Radioactive Nanoparticles for in Vivo Dual Mode Imaging.

Multimodal imaging agents combine two or more imaging modalities into one probe. Self-assembling fluorescent nanoparticles are a promising class of modular multimodal imaging probes as they can allow easy blending of imaging and targeting modalities. Our group recently developed a class of self-assembling and intrinsically fluorescent small molecule-based nanoparticles (SMNPs) with excellent optical properties. In this article, we describe the efficient radiolabeling of these SMNPs via a two-step bioconjugation strategy involving the inverse-electron-demand Diels-Alder ligation between a tetrazine (Tz)-tagged radiolabel and a trans-cyclooctene (TCO)-tagged fluorescent small molecule building block of the SMNPs. Studies in mice revealed that the SMNPs are well tolerated and could be monitored by both radioactivity and fluorescence, thereby demonstrating the potential of SMNPs in optical and dual-mode imaging in vivo. The work also testifies to the utility of the Tz-TCO conjugation chemistry for the labeling of self-assembled nanoparticles.

Author Correction: Chemically triggered drug release from an antibody-drug conjugate leads to potent antitumour activity in mice.

The original version of this Article omitted the following from the Acknowledgements: 'This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, through the Breast Cancer Research Program under Award No. W81XWH-15-1-0692. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense'. This error has now been corrected in the PDF and HTML versions of the Article.

Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes.

Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.

Chemically triggered drug release from an antibody-drug conjugate leads to potent antitumour activity in mice.

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.

Click-to-Release from trans-Cyclooctenes: Mechanistic Insights and Expansion of Scope from Established Carbamate to Remarkable Ether Cleavage.

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.

Fluorescence- and multispectral optoacoustic imaging for an optimized detection of deeply located tumors in an orthotopic mouse model of pancreatic carcinoma.

A crucial point for the management of pancreatic ductal adenocarcinoma (PDAC) is the decrease of R1 resections. Our aim was to evaluate the combination of multispectral optoacoustic tomography (MSOT) with fluorescence guided surgery (FGS) for diagnosis and perioperative detection of tumor nodules and resection margins in a xenotransplant mouse model of human pancreatic cancer. The peptide cRGD, conjugated with the near infrared fluorescent (NIRF) dye IRDye800CW and with a trans-cyclooctene (TCO) tag for future click chemistry (cRGD-800CW-TCO), was applied to PDAC bearing immunodeficient nude mice; 27 days after orthotopic transplantation of human AsPC-1 cells into the head of the pancreas, mice were injected with cRGD-800CW-TCO and imaged with fluorescence- and optoacoustic devices before and 2, 6 and 24 hr after injection, before they were sacrificed and dissected with a guidance of FGS imaging system. Fluorescence imaging of cRGD-800CW-TCO allowed detection of the tumor area but without information about the depth, whereas MSOT allowed high resolution 3 D identification of the tumor area, in particular of small tumor nodules. Highly sensitive delineation of tumor burden was achieved during FGS in all mice. Imaging of whole-mouse cryosections, histopathological analysis and NIRF microscopy confirmed the localization of cRGD-800CW-TCO within the tumor tissue. In principle, all imaging modalities applied here were able to detect PDAC in vivo. However, the combination of MSOT and FGS provided detailed spatial information of the signal and achieved a complete overview of the distribution and localization of cRGD-800CW-TCO within the tumor before and during surgical intervention.

Metal-Free Cycloaddition Chemistry Driven Pretargeted Radioimmunotherapy Using α-Particle Radiation.

The pretargeted radioimmunotherapy approach (PRIT) decouples the administration of tumor targeting monoclonal antibodies (mAbs) from that of the radiolabeled ligand. This multistep strategy allows delivery of high doses of radiation to tumor cells while minimizing nonspecific normal tissue irradiation. In this study, we evaluated the potential of pretargeted α-particle radioimmunotherapy based on the inverse electron demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). Two tetrazine based chelators, DOTA-Tz and TCMC-Tz, were synthesized and compared for their radiolabeling efficiency with 212Pb, radiochemical stability, and in vivo pharmacokinetics. Dosimetry was determined from pretargeted biodistribution studies. The PRIT study was carried out in LS174T tumor bearing mice pretargeted with CC49-TCO mAb. After removing unbound mAbs from the blood using two doses of clearing agent, mice were treated with various doses of (0, 2.78, 4.63, 7.40, and 2 × 2.78 MBq) of 212Pb-DOTA-Tz. 212Pb-DOTA-Tz displayed better in vivo biodistribution than 212Pb-TCMC-Tz and was selected for PRIT study. All the mouse groups receiving treatment displayed a dose dependent reduction in tumor size, while the control groups showed exponential tumor growth. Treatment with 2.78, 4.63, and 2 × 2.78 MBq of 212Pb-DOTA-Tz resulted in statistically significant improvement in median survival (26, 35, and 39 days, respectively). Groups receiving 7.40 MBq of 212Pb-DOTA-Tz and 0.55 MBq of direct labeled CC49 exhibited acute radiation associated toxicity. This study successfully demonstrated that pretargeted 212Pb α-particle therapy resulted in reduced tumor growth rates and improved survival with minimal normal tissue toxicity.

DOTA-tetrazine probes with modified linkers for tumor pretargeting.

INTRODUCTION: Pretargeted radioimmunoimaging and -therapy approaches building on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have yielded impressive results in recent years and have proven a vital alternative to biological pretargeting systems. After improvement of the TCO-antibody conjugates, we here report on our evaluation of a new series of radiolabeled Tz-probes. METHODS: Four new Tz-probes were synthesized, radiolabeled with lutetium-177, and characterized in vitro in terms of lipophilicity, reactivity, and stability in PBS and mouse serum. The in vivo biodistribution profile and tumor-targeting potential of the probes were evaluated in LS174T tumor-bearing mice pretargeted with TCO-antibody conjugates using non-pretargeted mice as control. RESULTS: Radiolabeling of all probes proceeded in high yields providing the 177Lu-labeled tetrazines in >95% radiochemical purity without any further purification. In mouse serum, half-lives of the probes varied between 8 and 13 h, with the exception of the most lipophilic probe, [177Lu]1b, with a serum half-life of less than 1 h. This probe also showed the fastest blood clearance (t1/2 = 5.4 min), more than 2-fold faster than PEG-linked probes [177Lu]3 and [177Lu]4, and even 3-fold faster than the other small probes without the PEG-linker, [177Lu]1a and [177Lu]2. In the pretargeting experiments, tumor uptake of the lead probe [177Lu]4 (~6 %ID/g) was most closely approached by [177Lu]2, followed by [177Lu]3 and [177Lu]1a. While all the smaller and more lipophilic probes suffered from increased liver uptake, the PEG-linked probe [177Lu]3 with its additional negative charge surprisingly showed the highest kidney uptake among all of the probes. CONCLUSION: The in vitro performance of some of the new tetrazine probes turned out to be comparable to the established lead probe [177Lu]Lu-DOTA-PEG11-Tz ([177Lu]4). However, tumor pretargeting studies in vivo showed lower tumor uptake and increased uptake in non-target organs.

Triggered Drug Release from an Antibody-Drug Conjugate Using Fast "Click-to-Release" Chemistry in 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.

Feasibility of Affibody-Based Bioorthogonal Chemistry-Mediated Radionuclide Pretargeting.

UNLABELLED: Affibody molecules constitute a new class of probes for radionuclide tumor targeting. The small size of Affibody molecules is favorable for rapid localization in tumors and clearance from circulation. However, high renal reabsorption of Affibody molecules prevents the use of residualizing radiometals, including several promising low-energy ß- and α-emitters, for radionuclide therapy. We tested a hypothesis that Affibody-based pretargeting mediated by a bioorthogonal interaction between trans-cyclooctene (TCO) and tetrazine would provide higher accumulation of radiometals in tumor xenografts than in the kidneys. METHODS: TCO was conjugated to the anti-human epidermal growth factor receptor 2 (HER2) Affibody molecule Z2395. DOTA-tetrazine was labeled with (111)In and (177)Lu. In vitro pretargeting was studied in HER2-expressing SKOV-3 and BT474 cell lines. In vivo studies were performed on BALB/C nu/nu mice bearing SKOV-3 xenografts. RESULTS: (125)I-Z2395-TCO bound specifically to HER2-expressing cells in vitro with an affinity of 45 ± 16 pM. (111)In-tetrazine bound specifically and selectively to Z2395-TCO pretreated cells. In vivo studies demonstrated HER2-specific (125)I-Z2395-TCO accumulation in xenografts. TCO-mediated (111)In-tetrazine localization was shown in tumors, when the radiolabeled tracer was injected 4 h after an injection of Z2395-TCO. At 1 h after injection, the tumor uptake of (111)In-tetrazine and (177)Lu-tetrazine was approximately 2-fold higher than the renal uptake. Pretargeting provided more than a 56-fold reduction of renal uptake of (111)In in comparison with direct targeting. CONCLUSION: The feasibility of Affibody-based bioorthogonal chemistry-mediated pretargeting was demonstrated. The use of pretargeting provides a substantial reduction of radiometal accumulation in kidneys, creating preconditions for palliative radionuclide therapy.