Thanasi, Ioanna Aikaterini;
(2025)
Enabling site-selective lysine modification on antibodies via proximity induced intramolecular reactivity.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Protein modification is a highly valuable and impactful tool for the development of bioconjugates, which offer significant potential in both diagnostic and therapeutic applications. Notably, and most directly related to this work, antibody and antibody fragment modification/bioconjugation has enabled crucial breakthroughs in cancer therapeutic treatments such as antibody-drug conjugates (ADCs). Over the past years, extensive research has been conducted for the development of methodologies for attaining efficient and selective antibody and/or antibody fragments bioconjugation. In particular, lysine modification is among the most widely employed strategies, primarily due to the generally high abundance of lysine residues on a protein’s surface and the nucleophilicity of their amine side-chains. Nevertheless, the vast number of solvent accessible lysine residues, which often exhibit comparable reactivity, poses a significant inherent challenge when aiming for regioselectivity and/or controlled loading of an entity. Therefore, research aimed at achieving the controlled loading of an entity via reaction with lysine residues is of great significance. This work focuses on the development of a novel strategy to achieve the controlled loading of an entity on lysine residues on a model native antibody Fab fragment. It was envisaged that this would be accomplished by exploiting the quantitative and reversible site-selective modification of disulfides using pyridazinedione reagents, which would enable near-quantitative proximity induced reactions with lysines. Through careful optimisation, outlined in the following sections, a novel strategy was developed allowing the formation of mono-labelled lysine modified antibody fragment conjugates via the formation of stable amide bonds. The strategy was appraised on several clinically relevant antibody fragments and click chemistry was employed to achieve modular modification. Furthermore, the formation of an orthogonally bis-labelled modified antibody fragment conjugate was accomplished via the usage of multiple cycles of this novel strategy.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Enabling site-selective lysine modification on antibodies via proximity induced intramolecular reactivity |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10204778 |
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| 2. |  Germany | 2 |
| 3. |  France | 1 |
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