Optimization of biorthogonal pretargeting for immuno-PET imaging in transgenic mouse models of Alzheimer's disease

Date: 10 November 2017

Venue: Campus Drie Eiken - Building Q - promotiezaal - Universiteitsplein 1 - 2610 Antwerp (Wilrijk) (route: UAntwerpen, Campus Drie Eiken)

Time: 4:00 PM - 6:00 PM

PhD candidate: Jens Fissers

Principal investigator: Koen Augustyns

Co-principal investigator: Leonie Wyffels

Short description: PhD defence Jens Fissers - Department of Pharmaceutical Sciences


The goal of this project was the evaluation of immuno-PET as a quantitative and sensitive in vivo imaging technique for non-invasive visualization of specific Amyloidß (Aß) and tau epitopes in transgenic mouse models with Alzheimer’s disease. Therefore, in-house J&J monoclonal antibodies (mAbs) against different epitopes in Aß and tau were assessed as potential PET-tracers using two different strategies.

First mAbs were directly radiolabeled with zirconium-89 (89Zr). 89Zr has good decay characteristics for optimal resolution and quantitative accuracy and has a physical half-life (t1/2 78h) compatible with the biological half life of mAbs. Anti-tau mAb [89Zr]-Df-AT8 demonstrated low in vivo stability and was therefore excluded from further in vivo evaluation. Anti-Ab mAbs [89Zr]-Df-Bz-JRF/AßN/25 and [89Zr]-Df-Bz-PRD/pE3/1 demonstrated higher stability, yet µPET imaging suggested limited utility of the 89Zr-labeled mAbs as amyloid imaging agents with the current methods due to non-specific binding and poor brain penetration of the immunoconjugates. Nevertheless, these radiotracers demonstrated in vivo plaque binding and strategies that increase brain penetrance would likely achieve better results.

One of the strategies to achieve immunoPET imaging of brain targets is to use inverse electron demand Diels-Alder (IEDDA) based bioorthogonal pretargeting. This strategy applies small molecule bioorthogonal tags (trans-cyclooctene (TCO) or tetrazine) that are radiolabeled with shorter living isotopes and which in vivo will bind to mAbs modified with the corresponding bioorthogonal tags (TCO or tetrazine). In this project mAbs were modified with tetrazines followed by detection with 18F/68Ga-labeled TCO.

We first synthesized a known 18F-labeled TCO to assess its usefulness for pretargeted immunoPET imaging in the brain. Evaluation of the ex vivo biodistribution and brain uptake revealed that [18F]TCO-1 enters the brain, but is quickly metabolized in vivo. We therefore developed new TCO analogues. Three novel 18F-labeled and one 68Ga-labeled TCO PET probe of which Al[18F]NOTA-TCO-5 showed promising imaging characteristics with high in vivo stability up to one-hour post injection and retained reactivity towards tetrazines. Simultaneously, new tetrazine analogues were synthesized with high in vitro stability and sufficient reactivity towards TCO. One tetrazine (3) was selected for coupling to trastuzumab, an anti-HER2/neu mAb chosen for a proof-of-concept study. In a final study we evaluated the pretargeted imaging capacity of our novel Al[18F]NOTA-TCO-5 PET-tracer and trastuzumab-tetrazine 3 in a HER2 positive xenograft mouse model. Unfortunately no tumor uptake could be demonstrated for Al[18F]NOTA-TCO-5 in a µPET imaging study. A combination of slow reaction kinetics, fast blood clearance, internalization of the mAb and lack of cell permeability of our TCO were probably the cause for this failure. Our pretargeting stratey needs further optimization to allow successful pretargeted immunoPET imaging.

Link: https://www.uantwerpen.be/en/faculties/fbd/research/departments-and-rese/department-of-pharma/