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Antibody Conjugates Via Disulfide Bridging: Elizabeth Ann Hull

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Antibody Conjugates Via Disulfide Bridging: Elizabeth Ann Hull Antibody Conjugates via Disulfide Bridging: Towards therapeutic and diagnostic applications Elizabeth Ann Hull A thesis submitted to University College London in accordance with the requirements of the degree of Doctor of Philosophy Supervisor: Dr James R Baker December 2014 1 I, Elizabeth Ann Hull confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. ……………………………….. 2 Abstract Antibodies play a prominent role in chemical and biological research and the largest application of chemical bioconjugation reagents is in the production of antibody conjugates. These conjugates provide a means of highly sensitive detection, for example in enzyme-linked immunosorbent assay (ELISA) systems. In therapeutics, such conjugates have enabled the development of bispecifics and antibody-enzyme directed prodrug therapy (ADEPT). Long-established chemical modification techniques for the conjugation of antibodies yield highly heterogeneous products. This heterogeneity is far from optimal and for therapeutic use antibody conjugates must be of a defined composition. Recently the site-specific introduction of chemical linkers has been reported through unnatural amino acid insertion. In this approach however, each protein must undergo successful mutation and expression prior to conjugation. To avoid this, an ideal site-directed conjugation technique would use residues natural to the protein. A new class of chemical bioconjugation reagents, the 3,4-substituted maleimides, allow the selective modification and bridging of naturally occurring protein disulfide bonds. In this thesis, the generation of homogeneous antibody-protein conjugates using 3,4- substituted maleimide based cross-linkers is investigated, with a focus on producing conjugates for ADEPT and bispecific therapeutics. A range of direct and indirect chemical cross-linking strategies via disulfide bridging are explored and the consequences of each approach examined. Ultimately, a new chemical platform to generate site-specific, homogeneous, antibody- antibody conjugates by targeting and bridging disulfide bonds was developed. A bispecific antibody construct was produced in good yield using a readily synthesised bis-dibromomaleimide cross-linker. Binding activity of antibodies was maintained, and in vitro binding of target antigens was observed. This technology is demonstrated through linking scFv and Fab antibody fragments, showing its potential for the construction of a diverse range of bispecifics. Finally, the ability of 3,4-substituted maleimide based reagents to functionalise antibodies for diagnostic applications is investigated. A strategy for the modification of a scFv-Fc construct with commercially available fluorophores is achieved and a synthetic route towards reagents suitable for immuno-PET applications determined. 3 For my parents, Martin & Karen Hull In loving memory of Arthur George Derek Godfrey 1930 - 2014 4 Acknowledgements I would like to start by thanking my supervisor, Dr James “Jamie” Baker. It has been a great pleasure to be part of his group for the past three (and a bit!) years. I feel very lucky to have had a supervisor who is so enthusiastic, encouraging and supportive, as well as a much needed calming influence at times! I would also like to thank my second supervisor, Professor Kerry Chester, for her constant encouragement and confidence boosting! I am grateful to Jamie and Kerry for the freedom they have given me over my PhD research and for the fantastic opportunity to learn a huge range of research skills. Thanks to Dr Abil Aliev for providing me with great guidance and support in everything NMR during my PhD and to Dr Lisa Harris, Dr Kersti Karu and Eifion Robinson for their excellent mass spectrometry advice and assistance. I would also like to acknowledge those who have provided additional supervision and support, namely my thesis chair Professor Elizabeth Shephard, Dr Mark Smith and Professor Stephen Caddick. I would particularly like to thank past and present members of the Baker group; Sally Fletcher, Rosemary Huckvale, Dr Favaad Iqbal, Dr Cristina Marculescu, Dr João Nunes, Daniel Richards, Dr Andrew Roupany and Dr Felix Schumacher. More recently, to Nafsika Forte and Dr Florian Kampmeier. Particular thanks to Felix, who patiently taught me the art of protein modification in those early days! I would like to thank many KLB 230 and 237 lab members; I have made many lasting friendships here and laughed a lot. Particular thanks to Dr Rhian Turner, Dr Vincent Gray, Samantha Gibson and Antoine Maruani. At the Cancer Institute I would like to thank the Chester group, who have dealt with my coming and going over the last three years! Particular thanks to Maria Livanos and Dr Enrique Miranda Rota, for their friendship and assistance to the “resident chemist”! I would like to say a special thanks to Sally Fletcher and Joanna Hemming. After meeting in the first week of our PhD programmes four years ago, we made a life-long friendship. Your support and presence throughout the PhD has been invaluable. 5 Finally, I would like to thank my family. My sister, Katherine, is endlessly supportive of my research career and always willing to provide scientific advice and assistance, and happily proof-read this thesis! My incredibly supportive and loving husband Anthony, who has successfully dealt with the emotional rollercoaster that is a PhD! His undoubting belief in my capabilities has given me the confidence to succeed. Most of all, I would like to thank my parents, to whom this thesis is dedicated. They have worked tirelessly to ensure my sister and I received a good education and a life full of opportunities. Without their unyielding support we would not be where we are today. 6 Contents Abstract…………………………………………………………………………………3 Acknowledgements……………………………………………………………………..5 Table of contents………………………………………………………………………..7 Abbreviations………………………………………………………………………….10 1 Introduction ............................................................................................................. 13 1.1 Antibodies ......................................................................................................... 13 1.2 Antibody Structure .......................................................................................... 13 1.3 Engineering Therapeutic Antibodies ............................................................. 15 1.4 Antibody fragments ......................................................................................... 16 1.4.1 scFv ............................................................................................................. 17 1.4.2 Extending fragment half-life ....................................................................... 18 1.5 Antibody conjugates for targeted therapeutics ............................................. 19 1.5.1 Direct targeting............................................................................................ 20 1.5.1.1 Radio-immunoconjugates ........................................................................ 20 1.5.1.2 Antibody-drug conjugates........................................................................ 20 1.5.1.3 Immunotoxins .......................................................................................... 21 1.5.2 Indirect targeting ......................................................................................... 21 1.5.2.1 ADEPT..................................................................................................... 22 1.6 Bispecific antibody therapeutics ..................................................................... 24 1.7 Antibody conjugates for diagnostics .............................................................. 27 1.7.1 Immunoassays ............................................................................................. 27 1.7.2 Immunohistochemistry ................................................................................ 28 1.7.3 Flow cytometry ........................................................................................... 29 1.7.4 Immuno-PET & Immuno-SPECT ............................................................... 30 1.8 Chemical modification of antibodies .............................................................. 31 1.8.1 Heterobifunctional cross-linkers ................................................................. 31 1.8.2 Site-directed conjugation ............................................................................ 36 1.8.2.1 Reduction of interchain disulfides ........................................................... 36 1.8.2.2 Oxidation of carbohydrate chains ............................................................ 38 1.8.2.3 Cysteine introduction by mutagenesis ..................................................... 38 1.8.2.4 Bioorthogonal reagents ............................................................................ 39 1.8.3 Bridging of disulfide bonds: a new class of reagents .................................. 46 1.9 Project aims ...................................................................................................... 57 2 Results and Discussion ............................................................................................ 61 2.1 Synthesis and application of a disulfide to lysine linker .............................. 61 2.1.1 Synthesis of dibromomaleimide linker ....................................................... 61 2.1.2
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