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Pierre Adumeau, René Raavé, Christian B. Jacobsen, Gerwin Sandker, Sandra Heskamp, Otto Boerman, Mark Rijpkema, Floriane Mangin, Michel Meyer, Jean-Claude Chambron, Mathieu Moreau, Claire Bernhard, Adrien Dubois, Laurène Da Costa, Victor Goncalves, Franck Denat

EMIM 2019, Glasgow, #654. EMIM 2019 Abstracts.

Abstract

Introduction

For immunoPET with ⁸⁹Zr, the current gold standard to label antibodies is desferrioxamine (DFO).1 However, preclinical studies have shown that the ⁸⁹Zr-DFO complex is partly unstable in vivo, leading to ⁸⁹Zr release and subsequent accumulation in mineral bone. This bone uptake may impede the detection of bone metastases, and hampers accurate estimation of the radiation dose to the bone marrow in dose planning for radioimmunotherapy. Therefore, there is a need for more stable ⁸⁹Zr chelators.

Methods

We have synthesized new octacoordinating ⁸⁹Zr-bifunctional chelating agents derivated from the DFO* chelator.² These new chelators were synthesized by coupling different hydroxamic acid-bearing arms to DFO, followed by the introduction of an isothiocyanate moiety. The model antibody trastuzumab was conjugated to the NCS-derivated chelators and DFO-pPhe-NCS as a reference, and radiolabeled with ⁸⁹Zr. The stability of the radiolabeled chelators and radiolabeled conjugates were evaluated in human plasma, and in PBS in presence of EDTA or DFO. The in vitro behavior of the most promising compounds was investigated more thoroughly using HER2-experessing SK-OV3 cells, and in vivo distribution was studied in mice with subcutaneous SK-OV3 xenografts by PET/CCT imaging and ex vivo tissue analysis.

Results/Discussion

The bifunctional chelators were conjugated efficiently to trastuzumab. Radiolabeling of the conjugates with ⁸⁹Zr yielded the radioconjugates with high yield, purity and specific activity (RCY >95%, RCP >99%, SA >100 MBq/mg). When challenged with EDTA or DFO, the ⁸⁹Zr-chelates and the corresponding radioconjugates displayed an improved stability compared to ⁸⁹Zr-DFO and ⁸⁹Zr-DFO-trastuzumab, with the best results obtained for the chelator dubbed cycloDFO*. The immunoreactive fraction and IC50 were similar for ⁸⁹Zr-DFO-trastuzumab and ⁸⁹Zr-cycloDFO*-trastuzumab. Internalisation after 2h was significantly higher for ⁸⁹Zr-cycloDFO*-trastuzumab compared to ⁸⁹Zr-DFO-trastuzumab. Accumulation of ⁸⁹Zr in bone was significantly lower for ⁸⁹Zr-DFO-cyclo*-trastuzumab compared to ⁸⁹Zr-DFO-trastuzumab in knee (3.6 ± 0.4% vs 5.9 ±0.6%), femur (2.2 ± 0.2% vs 3.4 ± 0.3%), and sternum (3.5± 0.4% vs 4.5 ±0.4%) at 72 h after injection. Uptake in the SK-OV3 tumor was similar for both antibody conjugates.

Conclusions

The new ⁸⁹Zr-chelators and the associated radioconjugates show improved in vitro stability compared to DFO and ⁸⁹Zr-DFO-trastuzumab. The radioconjugate derivated from the more promising chelator, ⁸⁹Zr-cycloDFO*-trastuzumab, demonstrated a better in vivo stability compared to ⁸⁹Zr-DFO-trastuzumab. Therefore, less radiation exposure to bone marrow and improved bone metastasis detection could be achieved using cycloDFO*.

References

¹ S. Heskamp, R. Raavé, O. Boerman, M. Rijpkema, V. Goncalves, F. Denat, Bioconjugate Chem., 2017, 28, 2211-2223.

² D. Vugts, C. Klaver, C. Sewing, A. Poot, K. Adamzek; S. Huegli, C. Mari, G. Visser, I. Valverde, G. Gasser, T. Mindt, G. van Dongen, Eur. J. Nucl. Med. Mol. Imaging, 2017, 44, 286-295