'Last step' enzymatic [18F]-labelling of peptides for Positron Emission Tomography (PET)

Lead Research Organisation: University of St Andrews
Department Name: Chemistry


Positron emission tomography (PET) is the most sensitive functional imaging method clinically and it application is growing rapidly through the Western world and developing countries, particularly as a diagnostic imaging tool for cancers and degenerative neurological disorders. Many major hospitals and clinical research centres in the Europe, the US and Asia are now commissioning cyclotrons and developing PET research facilities locally. Fluorine-18 is an important isotope for PET. It has a relatively long half-life (109 mins) and is readily generated in a cyclotron in the form of [18F]-fluoride ion, in very high specific activity (GBq's) from oxygen-18 water. As a consequence new methods to develop C-[18F]F bond formation for PET labelling are in demand, in general the link between fluorine chemistry and pharmaceutical/medical applications is strong. Approximately 20% of all pharmaceutical, since the 1950s, contain a fluorine atom and the bio-distribution of all new pharmaceutical products are required to be explored by PET, as part of clinical trials. Also new PET tracers are in demand as tools for early diagnosis as indicators of disease states. New fluorine chemistry is required to meet the demands of a growing and dynamic PET research community both in the UK and internationally. This proposal aims to develop a novel methodology for incorporating fluoride-18 specifically into peptides and proteins.

In this proposal we aim to exploit a novel enzyme which can form C-F bonds from fluoride ion. The fluorinase enzyme was discovered in 2002 (Nature, 2002, 416, 279) in St Andrews and it has been over-expressed and its structure (X-ray) and mechanism elucidated. The enzyme catalyses the reaction of fluoride ion and S-adenosyl-L-methionine (SAM) to generate 5'-FDA and L-methionine. It has proven to be a chemoselective biotransformation method for generating C-18F bonds from inorganic [18F]-fluoride. However we have found a weakness in the substrate specificity. We find that at a very specific location we can attach a linker to the substrate, and it will be accepted by the enze, this linker provides an anchor point to run a molecular line (poly ethylene glycol) to a peptide molecule of choice. The chosen peptides are those that identify cancer cells in the body, known as homing peptides, or small antibodies called 'affibodies' that identify tumour cells. In this way we can use the enzyme to attach the fluorine-18 isotope. The important advantage is that the fluoride-18 is generated in water, and the enzyme functions in water at neutral pH. Also peptides are nicely soluble in water, so the labelling can take place without the difficulty of using organic solvents for these biomolecules. This presents attractive possibilities. The fluorinase is the only example of an enzyme used in fluorine-18 PET synthesis and in this regard it offers an entirely new method for incorporating fluorine. In practical terms it has emerged to be particularly appropriate, because PET uses picomolar [18F]-fluoride ion, but the over-expressed fluorinase enzyme is present at mg/ml (microM), and therefore the kinetics favour C-18F synthesis due to a large molar excess of enzyme.

This is a research collaboration between the Universities of St Andrews and Aberdeen where the enzymatic methods for labelling the petides and proteins will be developed in St Andrews and the radiolabeling protocols carried out at the Aberdeen PET Centre, situated in the Aberdeen Royal Infirmary. The major focus of the research will concentrate on rapid labelling of peptides under neutral ambient conditions.

The research in aims to establish new methods for much wider applications by the growing international research community of PET radiochemists and we have ambitions to translate the methods to the clinic through interactions with PET based companies such an Imanova and our established interactions with the Beatson Cancer Institute in Glasgow.

Planned Impact

Who will benefit?
The research will develop a method for radiolabelling proteins and peptides for positron emission tomography (PET), in clinical imaging agents e.g. for cancer. Most importantly it will develop a method for the production of fluorine-18 labelled peptides and proteins, in water at neutral pH using the fluorinase enzyme. The fluorinase has the potential to be utilised by a variety of PET Centres and radio-chemists involved in protein labelling for imaging. These chemists work in PET radiochemistry labs closely associated with the pharmaceuticals industries or directly in clinical research environments. So the research has the potential to impact quickly and directly on translational medicine research environments. Outstanding success, perhaps in 10 years time would see fluorinase method being used internationally to prepare a labelled form of a specific peptide or protein, that will impact on disease diagnosis and contribute to improving health treatment. In that respect members of the public will benefit.

How will they benefit?
The programme will demonstrate the potential and range of the fluorinase methodology for the non-destructive labelling of peptides and proteins. It should extend the range of possibilities that can be developed, and in turn assist in the development of new radiotracers for diagnosis and monitoring disease progression.

What will be done?
The outcomes of the research will be published in leading journals, following the pattern from our recent research projects. The methodology and how to apply it will be available quickly to the international community. Patenting and IPR protection will be required for societal impact. This is already in place, but can be developed for individual applications, e.g. specific protein labelling. Such IPR will most reasonably be pursued at the individual research project level, as developments unfold. The important aspect of this programme is to demonstrate the scope of the methodology to the international community and demonstrate the potential that may lie in fluorinase biocatalysis applied to PET.

Methods of Communication and engagement
The research will be published. Presentations will be made at key international conferences both in fluorine/pharma chemistry environments and among the radiochemistry/PET Imaging community, following a current pattern by DO'H/MZ. Specific targeting of the imaging community will be pursued.
The PI and CoI are on the Executive Committee of SINAPSE (Scottish Imaging Network, a Platform for Excellence). ( http://www.sinapse.ac.uk/ )
They have access to major meetings and congregations of the International Clinical Imaging Community, through that mechanism. The SINAPSE connection enables an ongoing and intensive discussion with the relevant international community. SINAPSE are keen to project their successes and provide an excellent additional vehicle for dissemination. There is also a good connection to Imanova, a PET technology company supporting the pharmaceuticals industry.

Collaboration and exploitation
St Andrews has a full patent on the fluorinase (2002) which is maintained in key countries across the world. There is a sound platform on which to exploit any new technology. Through SINAPSE we have developed close working collaborations with Beatson Cancer Institute at the University of Glasgow, and Imanova based at the Hammersmith Hospital in London. Through such routes the impact of the research will reach front line translational medical laboratories working in the relevant area.
The growth of PET research centres now requires trained radiochemists with a specialist focus. This programme contributes directly to growing that skill base in the UK
Description In this grant we used a fluorination enzyme (fluorinase) to incorporate the fluorine-18 isotope into several radiotracers for utility in positron emission tomography. In particular we developed radiotracers for the PSMA receptor associated with prostrate cancer and also for A2A-adenosine receptors.
We were able to show that the fluorinase enzyme is an excellent catalyst for isotope incorporation, in water/buffer, for utility in this imaging modality.
The work form the grant had demonstrated the feasibility of the method for a range of different receptor targets. The challenge remains to progress this method to a clinical environment.
Exploitation Route They will be taken forward by continuing our interactions with chemists in the PET community. We continue our interactions with the PET centre at the University of Aberdeen.
Success here will require the identification of a particular tracer that the methodology is suited to, and that chemical methods are limiting.
Sectors Healthcare

Description EPSRC Responsive Mode
Amount £341,000 (GBP)
Funding ID EP/M01262X/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 02/2015 
End 01/2018
Description A2A receptor agonist labelling 
Organisation Leiden University
Country Netherlands 
Sector Academic/University 
PI Contribution We have collaborated with Leiden University where they have A2A receptor agonist assays
Collaborator Contribution Leiden were able to assay two novel seelctively fluorinated A2A receptor agonists prepare in our lab. These were also successfully labelled with fluorine-18 by out Aberdeen University collaborators, for positron emission tomography studies.
Impact A publication : Phillip T. Lowe, Sergio Dall'Angelo, Thea Mulder-Krieger, Adriaan P. IJzerman, Matteo Zanda and David O'Hagan, A novel class of fluorinated A2A adenosine receptor agonist with application to last step enzymatic [18F]fluorination for PET imaging, ChemBioChem., 2017, 18, 2156 - 2164 Multidicsiplinary - Chemistry and human biology
Start Year 2007
Description Aberdeen PET Centre 
Organisation University of Aberdeen
Department Health Services Research Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution We collaborate with Professor Matteo Zanda's group at the Positron Emission Tomography Centre at the Royal Infirmary, University of Aberdeen. They carry out the fluorine-18 experiments.
Collaborator Contribution Aberdeen PET Centre has been a medical imaging unit for over 20 years. It is now led by Professor Matteo Zanda an internationally recognised organo-fluorine chemist and fluorine-18 radiochemist. St Andrews and Aberdeen have collaborated closely applying fluorinase technology to PET. The PET laboratory at Abedeen has three radiochemists chemists who develop research programmes usually in conjunction with pharma. They host St Andrews scientists to carry out fluorinase catalysed radiolabelling experiments
Impact Funding and training of Scientists Ph.D student Stephen Thompson is now employed at the PET Centre at University of Michigan (Ann Arbour) Medical School
Start Year 2012