Chrzastek, Alina Anna; (2022) Dual reactivity disulfide bridging reagents for antibody bioconjugation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Antibody drug conjugates (ADCs) are monoclonal antibodies combined with drug moieties, these constructs jointly provide target specificity and potency. However, the majority of approved ADCs are heterogenous mixtures of species with different drug-to-antibody ratio (DAR) and conjugation sites, leading to species with varying toxicity profiles and pharmacokinetics. To overcome this problem, a major focus in the field is on creating homogenous ADCs. Approaches include engineering specific amino acid sequences that can be further used as a tag for enzymatic or chemical bioconjugation, however, these techniques are often laborious and costly. Disulfide rebridging also known as disulfide stapling, represents an intriguing new method for site-selective bioconjugation on native antibodies. This involves reduction of disulfide bonds, which are then reconnected by rebridging reagents, creating homogenous conjugates. In our research group, we have previously reported that bis-thioesters are disulfide rebridging reagents that can be further exploited to transfer to nearby lysines, offering site-selective lysine conjugation. In this work, further exploration of the thioester moiety has led to their application in creating multifunctional antibody fragment conjugates. A selection of novel bis-electrophiles containing the thioester moiety were synthesised and tested on a reduced antibody Fab fragment for their disulfide rebridging ability. It was found that thioester containing reagents can be designed and used to insert a ‘stable-labile’ linkage between the two cysteines. The reactive labile handle was employed in a subsequent bioconjugation step via native chemical ligation with an N-terminal cysteine peptide that allows for construction of antibody peptide conjugates. A second strategy was also developed, where use of hydrazine as a ligating nucleophile enabled attachment of two separate cargos on each Fab cysteine, which can be exploited to insert variably cleavable linkers. The insertion of a ‘stable- labile’ handle with thioesters has demonstrated an enticing new chemical toolbox to enable facile production of diverse antibody conjugates.

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