Abstract
Modern medicine uses molecular imaging techniques to study biological processes at the tissue and subcellular levels. With use of molecular imaging, cancerous lesions can be diagnosed, and aspects of disease progression can be monitored. Oncological imaging requires appropriate targeting of biomarkers and hallmarks of cancer. Harnessing the high specificity of monoclonal antibodies and antibody mimetics for diagnostic imaging purposes, allows for specifically targeted tumour uptake with high contrast over healthy tissues. This thesis explores the themes of conjugation chemistry and engineered protein constructs in the development of new molecular imaging probes.
Designed ankyrin repeat proteins (DARPins) are antibody mimics with a low molecular weight (15-20 kDa). Chapter 2 explores the use of radiolabelled DARPins conjugates as agents for imaging the overexpression of the HER2/neu receptor in ovarian and breast cancer. Both site-specific maleimide and sortase-A mediated transpeptidations were investigated as routes towards DARPin conjugates. With use of the enzymatic transpeptidation, a DFO-DARPin conjugate was synthesised. Radiolabelling of this conjugate with 89Zr allowed assessment of the tracer in HER2 expressing cell lines and xenografts. This radiotracer specifically accumulates in HER2 positive xenografts, albeit with high kidney uptake which is characteristic of these small repeat proteins. With exception of the kidneys, excellent tumour-to-tissue contrast was observed, and DARPins represent a promising protein scaffold for use in nuclear imaging.
Chapters 3 and 4 explore light induced protein ligations using aryl azide (ArN3) photoactive groups. In Chapter 3, a novel HBED-CC chelate derivative featuring an ArN3 functionality was synthesised. Two- step photochemical conjugation of this derivative to the engineered c-MET targeting monovalent antibody, MetMAb, and 68Ga radiolabelling resulted in the production of [68Ga]GaHBED-CC-azepin- MetMAb. This radiotracer was evaluated with stability studies, in vitro binding assays and PET imaging in vivo. The synthesised radiotracer could specifically delineate high levels of c-MET expression and accumulated in MKN-45 tumour tissues at 6 h post-administration. Building on this photochemical approach, in Chapter 4 a photoactivatable BODIPY probe bearing the ArN3 moiety was developed. Photochemical ligations of MetMAb and HSA with this BODIPY probe proceeded with high efficiency and gave fluorescent protein conjugates. These conjugates were characterised by using UV-Vis spectroscopy, SEC, and SDS-PAGE with fluorescent imaging.
Chapter 5 investigates the use of the fluorogenic photoclick reaction between functionalised tetrazoles and styryl derivatives. The aim of this work was to provide access to dual modality PET/OFI agents. In this chapter, bioconjugation experiments were performed to form styryl functionalised MetMAb. Photoclick reactions of this modified protein with a NODAGA functionalised tetrazole gave rise to fluorescent conjugates that were radiolabelled with 68Ga. During this work, we discovered that native, non-functionalised MetMAb could also be labelled with the NODAGA functionalised tetrazole, and therefore, the research plan was changed and functionalised tetrazoles were investigated for direct photo-ligation of protein scaffolds in Chapter 6.
In Chapter 6, a small synthetic screening of tetrazole analogues was performed and scaffolds were identified which could be photoactivated in the near-visible region to minimise UVB damage to biomolecules. Further functionalisation of a tetrazole analogue substituted with 4-methoxy
naphthylene provided access to NODAGA and DFO derivatives. These derivatives could ligate various proteins under 365/395 nm photoactivation. Ligation of trastuzumab with the DFO functionalised tetrazole and subsequent 89Zr radiolabelling led to the synthesis of a new radiotracer for HER2/neu PET imaging. This radiotracer was found to exhibit high radiochemical stability and excellent immunoreactivity in SK-OV-3 cells. The integrity of this radiotracer was assessed in vivo where high tumour uptake in SK-OV-3 xenografts was observed, and biodistribution profiles obtained at 72 h post administration were comparable to those obtained with [89Zr]Zr-trastuzumab-DFO conjugates synthesised through conventional strategies.
Overall, this thesis explores the use of conjugation chemistry and engineered protein scaffolds to deliver novel protein probes which have exhibited great promise for potential applications in nuclear medicine and optical imaging.
Fay, Rachael