Development of novel chemical tools for bioorthogonal pretargeted PET imaging

Datum: 30 maart 2020

Locatie: Campus Drie Eiken - Gebouw R - Auditorium R1 - Universiteitsplein 1 - 2610 Wilrijk

Tijdstip: 17 - 19 uur

Promovendus: Eduardo Figueiredo Pires Ruivo

Promotor: Koen Augustyns - Pieter Van der Veken

Korte beschrijving: Doctoraatsverdediging Eduardo Figueiredo Pires Ruivo - Departement Farmaceutische Wetenschappen


Pretargeted in vivo imaging using the bioorthogonal IEDDA reaction between TCO and Tz holds promise to allow imaging of mAbs with shorter living isotopes, to reduce radiation burden to the patient. This strategy can allow the development of a single radiolabeled probe that can be used with many mAbs, facilitating its use in R&D and clinical practice. For application of the TCO-Tz ligation, the radiolabeled probe should be stable and have fast reaction kinetics allowing reaction with the tag within minutes at low μM concentration.

The two-step in vivo labeling approach also facilitates the use of shorter living radioisotopes like 18F (T1/2 = 110 min) that would otherwise not be compatible with the long circulation times of high molecular weight molecules (mAb). Considerable research has been devoted to the development of mAb-TCO conjugates, and their application for pretargeted tumor imaging has been reported using diverse 18F-labelled tetrazines. However, it has been shown that TCO has the tendency to isomerize to its isomer, cis-cyclooctene (CCO), that is several orders of magnitude less reactive with tetrazine after prolonged exposure to physiological conditions.

Therefore, in this thesis we explored the inverse approach where a mAb is modified with tetrazine instead of TCO. A range of tetrazines was developed and evaluated regarding their stability and reactivity, and the most promising tetrazine was conjugated to mAb trastuzumab. In a proof of concept study we demonstrated the usefulness of the tetrazine-mAb conjugate for bioorthogonal pretargeted imaging using a live-cell fluorescent imaging experiment, using a TCO-fluorescent probe. Furthermore, we envisaged the translation of this strategy for in vivo pretargeted PET imaging applications. For this, a novel Al18F-NOTA labeled TCO radioligand was developed as a potential counterpart for IEDDA reaction with a tetrazine-tagged antibody.

The radiotracer showed improved in vivo metabolic stability (51.9 ± 5.16% after 1 h) compared to previous reported 18F-labeled TCOs, allowing a clear visualization of tumor tissue in a small-animal pretargeted PET imaging experiment. In an effort to increased absolute tumor uptake, we further developed next-generation TCO derivatives. The novel 18F-labeled dTCO-amide probe showed an extremely fast kinetic rate (10,553 M-1s-1 in 50:50 methanol:water), good stability in saline and plasma up to 4h at 37°C and a favorable biodistribution in healthy mice. Pretargeted µPET imaging experiments in mice bearing LS174T colorectal tumors, previously treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49), showed clear visualisation of tumor tissue with a significant higher uptake when compared to the control.