Towards the Dual Click ¹⁸F-labeling of Antibodies

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 naïve 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. Keinänen 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.

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