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Pierre Adumeau, René Raavé, Milou Boswinkel, Sandra Heskamp, Mathieu Moreau, Claire Bernhard, Laurène Da Costa, Victor Goncalves, Franck Denat

EMIM Conference 2020

Abstract

Introduction 
Immuno-PET/NIRF imaging is very promising for cancer therapy, as it allows non-invasive localization of the tumor and its image-guided resection. The mostly used strategy to synthesize such dual-labeled conjugates relies on a double, sequential random conjugation of the fluorophore and the radionuclide/chelator with the antibody. However, the random conjugation leads to high heterogeneity and potential loss of bioactivity and these phenomena are exponentially amplified by sequential modifications. Therefore, there is a need for a better dual-labeling strategy for PET/NIRF imaging.

Results/Discussion 
The trivalent platform BCN-DFO-IR800 was obtained in a five steps synthetic route with a global yield of 5%. Trastuzumab-N3, obtained through chemoenzymatic glycoengineering, was efficiently conjugated with the trivalent platform, leading to trastss-DFO/IR800 with a degree of labeling (DOL) of 2.0 (theoretical maximum). Trastrd-DFO/IR800 was synthesized with comparable DOL for the sake of comparison.
Radiolabeling of the conjugates with ⁸⁹Zr yielded the radioconjugates with high yield, purity and specific activity (RCY >95%, RCP >99%, SA >50 MBq/mg).
The site-specific conjugate displayed lesser aggregation over time than its random cousin (after 7 days in PBS: 5.0±0.1 % vs 12.7±5.2 % for trastss-DFO/IR800 and trastrd-DFO/IR800, respectively). Fluorescence intensity of the site-specific conjugate also showed an improved stability compared to the random conjugate, the first displaying 90±1 % of the initial fluorescence intensity after 7 days in PBS, with only 25±3 % for trastrd-DFO/IR800.

Conclusion 
This is the first example of a platform-based, site-specific PET/NIRF conjugate. This strategy gives complete control over the dual-labeling of antibody. The preliminary results have demonstrated the in vitro superiority of our conjugate over the classical random bimodal conjugate. We expect these results to translate into a superior in vivo behavior of the site-specific conjugate. In vivo experiment results will be presented at the conference.