TY - JOUR T1 - Development of <em>Trans­-</em>Cyclooctene Modified Antibodies for Pretargeted Imaging within the Central Nervous System JF - Journal of Nuclear Medicine JO - J Nucl Med SP - 196 LP - 196 VL - 61 IS - supplement 1 AU - Sara Lopes van den Broek AU - Vladimir Shalgunov AU - Dag Sehlin AU - Stina Syvanen AU - Matthias Herth Y1 - 2020/05/01 UR - http://jnm.snmjournals.org/content/61/supplement_1/196.abstract N2 - 196Introduction: Antibodies are highly selective and of high affinity towards their target, giving them the potential to provide high quality Positron Emission Tomography (PET) images when used as a radiotracer [1]. However, the use of antibodies as PET tracers for targets within the brain is challenging due to poor and slow brain penetration, requiring the use of long-lived isotopes when conventional imaging methods are used [2]. Pretargeted imaging is a two-step approach, where a tagged large molecule, e.g. an antibody, is administered first, allowed to accumulate at its target and then followed by administration of a second, much smaller and radiolabeled compound, which is usually administered after days (Figure 1). These two moieties react in vivo through bioorthogonal chemistry, allowing the use of short-lived isotopes, e.g. fluorine-18. The tetrazine ligation between an electron-poor tetrazine and an electron-rich trans cyclooctene moiety is a highly suitable bioorthogonal chemistry approach for pretargeting due to its high reaction kinetics and selectivity (Figure 2) [3]. In this study, trans-cyclooctene (TCO) modified antibodies were developed and applied to pretargeted in vitro autoradiography to estimate the minimal TCO concentration needed to achieve sufficient imaging contrast within the brain for future pretargeted in vivo studies. Antibodies targeted amyloid beta (Aβ). Methods: Monoclonal antibodies (mAbs) were modified using 100, 500, and 1000 eq. of TCO-PEG4-NHS. The number of reactive TCOs was determined by SDS-PAGE using a highly polar tetrazine labelled with indium-111. Affinity of the TCO-mAb conjugates towards their target was measured by ELISA and compared to the non-modified antibody. Autoradiography was performed on brain sections from transgenic mice (tg-ArcSwe), and wildtype mice using different TCO-mAb concentrations, where the cortex was used as the region of interest (high Aβ deposition) and the cerebellum as a reference region (low Aβ deposition). Figure 3 shows a schematic overview of the autoradiography methodology. Imaging was performed using the same 111In-Tz as mentioned before. Autoradiography results were analyzed using OptiQuant and quantified using ImageJ and Excel. Results: Modifications of the mAbs with 100, 500, and 1000 eq. of TCO resulted in mAb-TCO conjugates with 7, 18, and 19 TCOs/mAb respectively. Titration by SDS-PAGE showed that a maximum number of around ~19 accessible TCOs/mAb could be reached (Figure 4). This modification did not alter the affinity towards Aβ drastically when 100 or 500 eq. were used. In contrast, 1000 eq. of TCO did significantly reduce the affinity. Consequently, these mAbs were not used for further studies. Quantitative autoradiography showed shallow imaging contrast at a concentration of 0.006 μg/mL TCO-mAb and a pronounced imaging contrast at a concentration of 0.06 μg/mL TCO-mAb for the antibodies with 7 and 18 TCOs/mAb (Figure 5). Conclusions: Using mAbs with 7-18 TCO/mAb, we estimated the TCO-mAb concentration needed to achieve reasonable imaging contrast for pretargeted strategies to be not higher than 0.06 μg/mL TCO-mAb. Future studies are directed to transport the necessary amount of TCO-modified mAb into mice brains. Acknowledgements: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813528.Figure 1: Schematic overview of pretargeted imaging using an antibodyFigure 2: Tetrazine ligation by the Inverse Electron Demand Diels-Alder reactionFigure 3: 111In-Tz Autoradiography work-flowFigure 4: Number of TCOs per antibody Figure 5: Example of autoradiography images and quantification obtained using the TCO-mAb conjugates ER -