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Title: Antibody conjugates via disulfide bridging : towards therapeutic and diagnostic applications
Author: Hull, E. A.
ISNI:       0000 0004 5364 8994
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2015
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available