This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ New approaches to radionuclide imaging of cancer with gallium-68 Imberti, Cinzia Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 05. Oct. 2021 New approaches to radionuclide imaging of cancer with gallium-68 A thesis submitted by Cinzia Imberti In partial fulfilment of the requirements for the degree of Doctor of Philosophy University of London 2018 School of Biomedical Engineering and Imaging Sciences King’s College London 1 Abstract The positron emitting isotope 68Ga is gaining increasing interest in the radionuclide imaging community, due to its favourable decay properties and availability from a generator. Incorporation of 68Ga into biomolecules requires conjugation to a bifunctional chelator. The tris(hydroxypyridinone) ligand THPMe is a valuable chelator for 68Ga, being able to coordinate Ga(III) effectively in extremely mild conditions. These outstanding radiolabelling properties prompted further investigation of THPMe, as a prototype to develop improved tris(hydroxypyridinone) chelators, and as a tool to explore new strategies for cancer imaging with 68Ga. In the first part of this project, a new tris(hydroxypyridinone) chelator, THPH, was developed, where the N1-methyl group in the hydroxypyridinone ring was replaced by hydrogen. A new synthetic strategy was developed to achieve this. Quantitative 68Ga labelling of THPH was achieved in mild conditions and in vivo stability of the radiolabelled complex demonstrated. Spectrophotometric titration and competition experiments revealed preference for Ga(III) over Fe(III) for both THPMe and THPH, with the latter possessing higher Ga(III) selectivity. A library of THP derivatives is potentially available from an intermediate in THPH synthesis as a result of the new strategy. In a second part of this work, the ability of THPMe to interfere with 68Ga trafficking in biological systems was investigated. THPMe (24 nmol) was found able to chelate 68Ga in vivo, significantly accelerating its renal excretion in mice. We therefore explored use of THPMe as a blood clearance agent, to increase the tumour-to-blood activity ratio in the context of cancer imaging with 68Ga salts. Initial evaluation of THPMe in a melanoma model lead to inconclusive results, due to inconsistency of 68Ga-acetate biodistribution. However, successive evaluation in a colon cancer xenograft showed a three-fold increase in tumour-to-blood ratio for mice treated with THPMe compared to a control group, demonstrating THPMe potential as a blood clearance agent in this model. Finally, a new pretargeting strategy for 68Ga imaging with antibodies was investigated, based on the in vivo chelation of 68Ga(III) by a pretargeted THPMe-antibody conjugate. Two THPMe-antibody conjugates were prepared by attachment of THPMe derivatives to non-internalising antibodies and their favourable 68Ga labelling properties and affinity for their target antigen verified. However, in vitro/in vivo evaluation, showed no pretargeting ability for either of these compounds, likely due to a low availability of THPMe-antibody conjugates at the antigen site and to competitive binding of 68Ga by transferrin or other proteins. 2 Declaration I Cinzia Imberti confirm that no part of this thesis has been submitted in support of any other application for a degree or qualification of King’s College London, or any other university or institute of learning. I confirm that this work is my own. Where information has been derived from other sources it has been indicated in this thesis. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. 3 Acknowledgments As any journey worth its name, this 4-years-long adventure would not have been possible without the help and the support of an incredible number of people, both inside and outside KCL. The first huge thank you goes to my supervisor, Professor Phil Blower, who over the years has been able to nurture not only my passion for chemistry and science in general, but also my vocation to be an academic researcher. Thank you for teaching me how to harness my enthusiasm and creativity with method and scientific rigour. Thank you for trusting me enough to give me the freedom to explore different ideas and to make my own mistakes and learn from them. Thank you finally for your efforts to create such a collaborative and friendly environment in the department. A warm thank you to my second supervisor Dr Sophia Karagiannis, who has always been extremely supportive and helpful, and to her research group for the A375 cell line and the advice in establishing the xenograft model. I would like to thank Prof. Jason Lewis and Dr Brian Zeglis, who kindly hosted me in their laboratories at Memorial Sloan Kettering Cancer Centre and Hunter College. Being able to work with your groups was a great privilege. Thank you also to everyone I have pestered during my stay, especially to Pierre for taking such a good care of me despite my ridiculously absent mind (among lost scarves, and forgotten lab books I have lost count!). Thanks to Kelly, Kishore, Delphine & Pierre and Sophie & Yorann for making me feel welcomed from day 1! Thank you to all the other collaborators I had the luck to work with during these years. Thanks to Dr Enrique Miranda Rota and Prof Kerry Chester for providing the ANTI-CEA antibody used in these studies and to Dr Mauricio Morais, for the advice and help on site-specific conjugation and for witnessing the atrocious experience of my first SDS- PAGE without losing sympathy for me. A special thank you to Professor Bob Hider, whose chemical knowledge extends beyond the limits of my imagination. Thanks for all the inspiring discussion and advices and for adopting me in your wonderful group. Thanks also to Dr Yu-Lin Chen for introducing me to the fine art of metal titration and for always manage to make me laugh. Thanks to Dr Vincenzo Abbate for the extremely valuable advices and help throughout these years. Thanks also to Dr Agostino Ciribizzi and Siham Memdouh and all the rest of the FWB people for your chemistry help and advice and for your friendship. 4 Thank you to all the people in KCL that helped me in these years, thanks to Dr Kavitha Sunassee, Dr Jayanta Bordoloy, Dr Julia Baguna Torres and Dr Nisha K. Ramakrishnan for the help with the preclinical facility. Thanks to Soba Akinwunmi, Davide Poccecai and the rest of the IT team for being so efficient and friendly! Thanks to Barry Crook, Steve Catchpole and David Thakor for their excellent lab management. Thank you, David, also for all the fun (and foolishness) in the lab all these years, I think that department without you would be a gloomy place to work in! Thanks to all the PhD, master and undergraduate students that I had the pleasure to work with during these years. Thank you Maria, Calum, Catherine, Yifu, Tasnim, Joanna and Fahad for all your enthusiasm and passion, I feel honoured to have been able to help you during your projects. Thank you Jen for being an amazing lab buddy during these years, despite both of us being determined and competitive we have always managed to work nicely as a team (may be because you are ESTJ and I am ENTP?!). I could not have asked for a better companion for this journey! A special thank you to all the postdocs who have held my hand in the lab in these years. Thank you for the enthusiasm, passion and tenacity that you were able to infect me with. In particular, thanks to Dr Brett Paterson for all the chemistry tips and the lifelong lesson that I can survive without a mass spectrometer (TLC for life!)! Thanks to Dr Samantha Terry, for teaching me to love biology (almost) as much as chemistry, for introducing me to the magic world of radiobiology and last, but not least, for supporting me in these last few months of writing! Thank you to Dr Julia Blower for your tireless help with all sorts of experiments, for all the early mornings and late evenings together, and for teaching me some of my favourite English terms (which would not be appropriate to write in these thesis)! Special thanks to Dr Levente Meszaros who, despite stubbornly refusing to be my postdoc, has been giving me so many useful advices (and thesis writing reminders!) to greatly make up for it. The biggest of the thank you though goes to Dr Michelle Ma for all the incredible help, support and friendship during these four years.