Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors

Abstract

Because positron emission tomography (PET) and optical imaging are very complementary, the combination of these two imaging modalities is very enticing in the oncology field. Such bimodal imaging generally relies on imaging agents bearing two different imaging reporters. In the bioconjugation field, this is mainly performed by successive random conjugations of the two reporters on the protein vector, but these random conjugations can alter the vector properties. In this study, we aimed at abrogating the heterogeneity of the bimodal imaging immunoconjugate and mitigating the impact of multiple random. A trivalent platform bearing a DFO chelator for 89 Zr labeling, a NIR fluorophore -IRDye800CWand a bioconjugation handle was synthesized. This bimodal probe was site-specifically grafted to trastuzumab via glycan engineering. This new bimodal immunoconjugate was then investigated in terms of radiochemistry, in vitro and in vivo, and compared to the clinically relevant random equivalent. In vitro and in vivo, our strategy provides several improvements over the current clinical standard. The combination of site-specific conjugation with the monomolecular platform reduced the heterogeneity of the final immunoconjugate, improved the resistance of the fluorophore toward radiobleaching and reduced the non-specific uptake in the spleen and liver compared to the standard random immunoconjugate. To conclude, the strategy developed is very promising for the synthesis of better defined dual labeled immunoconjugates, although there is still room for improvement. Importantly, this conjugation strategy is highly modular and could be used for the synthesis of a wide range of dual labeled immunoconjugates.

A glyco-engineering approach for site-specific conjugation to Fab glycans

“…For instance, Adumeau et al have synthesized a trivalent imaging probe bearing a near-infrared fluorophore, a radionuclide-chelating agent and an azide-reactive bicyclononyne that was conjugated to trastuzumab that had an azidosugar attached to the Fc glycan. 19 However, our method would allow for double-labeling of the same antibody at different specific sites, which could be advantageous for conjugating distinct payloads that are bulky or hydrophobic, or that cannot be directly attached the same backbone molecule. Another site-specific chemoenzymatic method was reported to be able to remodel separately the Fab and Fc N -glycans of cetuximab.…”

First Comparison Study of the In Vitro and In Vivo Properties of a Randomly and Site-Specifically Conjugated SPECT/NIRF Monomolecular Multimodal Imaging Probe (MOMIP) Based on an aza-BODIPY Fluorophore

“…15,23−25 In particular, a study has shown that site-specific bioconjugation of a MOMIP IR Dye-DFO (desferrioxamine chelator) probe to a trastuzumab antibody had a very positive impact compared with random and sequential conjugation of the two probes, both on the stability of the resulting bioconjugates and on their biodistribution. 15 Regarding the off-target uptake, another study has highlighted the implication of FcγRI binding in the accumulation of antibodies in the liver and spleen and has shown that it could be attenuated via the truncation of the glycans on the heavy chains (via EndoS) in this site-specific approach. 23 In order to really evaluate the effect of deglycosylation via EndoS, we decided to compare EndoS-treated Wazaby-DOTA-rl-Trastu to Wazaby-DOTA-ss-Trastu.…”

Targeted Dual-Modal PET/SPECT-NIR Imaging: From Building Blocks and Construction Strategies to Applications

“…One clear trend is that mAb-targeting agents generally use class III “double-labeling” strategies. One emerging area is the development of homogenous protein labeling methods, which has been investigated for both pretargeting and direct dual labeling [ 43 , 44 , 45 ]. Going forward, there is significant potential to apply class I, II, and IV strategies to antibody and protein targeting agents, which may also benefit from homogenous bioconjugation approaches.…”