Pretargeted nuclear imaging for the diagnosis of various cancers is an emerging and fast developing field. The tetrazine ligation is currently considered the most promising reaction in this respect. Monoclonal antibodies are often the preferred choice as pretargeting vector due to their outstanding targeting properties. In this work, we evaluated the performance of [64Cu]Cu-NOTA-PEG7-H-Tz using a setup we previously used for [111In]In-DOTA-PEG11-BisPy-Tz, thereby allowing for comparison of the performance of these two promising pretargeting imaging agents. The evaluation included a comparison of the physicochemical properties of the compounds and their performance in an ex vivo blocking assay. Finally, [64Cu]Cu-NOTA-PEG7-H-Tz was evaluated in a pretargeted imaging study and compared to [111In]In-DOTA-PEG11-BisPy-Tz. Despite minor differences, this study indicated that both evaluated tetrazines are equally suited for pretargeted imaging.
1144 Objectives: Monoclonal antibodies (mAbs) are promising targeting-vectors for cancer diagnosis using nuclear molecular imaging techniques e.g. positron emission tomography (PET) due to their high affinity and specificity. However, their slow pharmacokinetics requires the use of long-lived radioisotopes in order to image their target accumulation. This results in high absorbed radiation doses for patients and a low imaging contrast.1,2 A pretargeted imaging approach, in which the targeting step is separated from the imaging step, circumvents these limitations since it enables the use of a short-lived radioisotope. This is achievable by using bioorthogonal click chemistry.3,4 Herein, we describe a pretargeted tumor imaging approach based on the inverse electron-demand Diels-Alder [4+2] cycloaddition (IEDDA) between various 18F-labeled 1,2,4,5-tetrazines and a mAb modified with trans-cyclooctene (TCO) (Figure 1). The sensitivity of the tetrazine-scaffold towards the basic conditions required for direct 18F-fluorination encouraged the use of an indirect labeling strategy by which a small library of 18F-tetrazines could be accessible for pretargeted imaging using PET. Methods: [18F]Fluoride was produced via the (p,n)-reaction by bombardment of a [18O]H2O with a 11 MeV proton beam in a cyclotron. Analysis of 18F-fluorination reactions were performed by radio-HPLC and radio-TLC. Small animal PET/CT imaging was performed in naive BALB/c mice for the preliminary biodistribution studies and in vivo stability assessments, and in nude BALB/c mice bearing tumor xenografts (LS174T) for the pretargeted tumor imaging experiments. Imaging was performed 1 h after tracer administration. Results: Tetrazine precursors bearing alkyne-moieties were synthesized and 18F-labeled with three different [18F]F-azide synthons via Cu-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) in non-decay corrected radiochemical yields between 2-22% for the CuAAC and 1-8% with regard to starting amount of [18F]F-. Promising candidates were selected based on biodistribution and stability for further evaluation in pretargeted experiments. For the pretargeted tumor imaging, TCO-functionalized CC49-mAbs targeting TAG-72 antigen were injected intravenously into tumor bearing mice 72 h prior to administration of high molar activity [18F]F-tetrazines. Preliminary results indicated a tumor uptake between 2-3% ID/g for the tetrazines evaluated until this date. Conclusions: By using the CuAAC as an indirect labeling approach a small library of 18F- labeled tetrazines was available. Evaluation in pretargeted experiments is still ongoing. Future work is to develop a group of second generation tetrazines with more favorable pharmacokinetic profiles. Acknowledgements: The authors greatly acknowledge the H2020 project Click-it for financial support and the technical staff at the Department of Clinical Physiology, Nuclear Medicine & PET at Rigshospitalet, Denmark. References: 1. Rossin R. et al., Angew. Chem. Int. Ed. 2010; 49. 2. Keinanen O. et al., EJNMMI Res. 2017; 7:95. 3. Herth M. M. et al., Chem. Commun. 2013; 49. 4. Denk C. et al., Angew. Chem. Int. Ed. 2014; 53.
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 logD7.4 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.
Purpose: Photothermal therapy (PTT) exploits the light-absorbing properties of nanomaterials such as silica-gold nanoshells (NS) to inflict tumor death through local hyperthermia. However, in in vivo studies of PTT, the heat distribution is often found to be heterogeneous throughout the tumor volume, which leaves parts of the tumor untreated and impairs the overall treatment outcome. As this challenges PTT as a one-dose therapy, this study here investigates if giving the treatment repeatedly, ie, fractionated PTT, increases the efficacy in mice bearing subcutaneous tumors. Methods: The NS heating properties were first optimized in vitro and in vivo. Two fractionated PTT protocols, consisting of two and four laser treatments, respectively, were developed and applied in a murine subcutaneous colorectal tumor model. The efficacy of the two fractionated protocols was evaluated both by longitudinal monitoring of tumor growth and, at an early time point, by positron emission tomography (PET) imaging of 18 F-labeled glucose analog 18 F-FDG. Results: Overall, there were no significant differences in tumor growth and survival between groups of mice receiving single-dose PTT and fractionated PTT in our study. Nonetheless, some animals did experience inhibited tumor growth or even complete tumor disappearance due to fractionated PTT, and these animals also showed a significant decrease in tumor uptake of 18 F-FDG after therapy. Conclusion: This study only found an effect of giving PTT to tumors in fractions compared to a single-dose approach in a few animals. However, many factors can affect the outcome of PTT, and reliable tools for optimization of treatment protocol are needed. Despite the modest treatment effect, our results indicate that 18 F-FDG PET/CT imaging can be useful to guide the number of treatment sessions necessary. Keywords: hyperthermia, cancer, nanoparticle, photothermal therapy, fractionated therapy, positron emission tomography
Pretargeted bioorthogonal imaging can be used to visualize and quantify slow accumulating targeting vectors with short-lived radionuclides such as fluorine-18 - the most clinically applied Positron Emission Tomography (PET) radionuclide. Pretargeting results in higher target-to-background ratios compared to conventional imaging approaches using long-lived radionuclides. Currently, the tetrazine ligation is the most popular bioorthogonal reaction for pretargeted imaging, but a direct18 F-labeling strategy for highly reactive tetrazines, which would be highly beneficial if not essential for clinical translation, has thus far not been reported. In this work, a simple, scalable and reliable direct 18 F-labeling procedure has been developed and applied to obtain a pretargeting tetrazine-based imaging agent with favorable characteristics (target-to-background ratios and clearance) that may qualify it for future clinical translation.
Peptide receptor radionuclide therapy (PRRT) relies on α- and β-emitting radionuclides bound to a peptide that commonly targets somatostatin receptors (SSTRs) for the localized killing of tumors through ionizing radiation. A Lutetium-177 (177Lu)-based probe linked to the somatostatin analog octreotate ([177Lu]Lu-DOTA-TATE) is approved for the treatment of certain SSTR-expressing tumors and has been shown to improve survival. However, a limiting factor of PRRT is the potential toxicity derived from the high doses needed to kill the tumor. This could be circumvented by combining PRRT with other treatments for an enhanced anti-tumor effect. Photothermal therapy (PTT) relies on nanoparticle-induced hyperthermia for cancer treatment and could be a useful add-on to PRRT. Here, we investigate a strategy combining [177Lu]Lu-DOTA-TATE PRRT and nanoshell (NS)-based PTT for the treatment of SSTR-expressing small-cell lung tumors in mice. Our results showed that the combination treatment improved survival compared to PRRT alone, but only when PTT was performed one day after [177Lu]Lu-DOTA-TATE injection (one of the timepoints examined), showcasing the effect of treatment timing in relation to outcome. Furthermore, the combination treatment was well-tolerated in the mice. This indicates that strategies involving NS-based PTT as an add-on to PRRT could be promising and should be investigated further.
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