Novel disulfide-bridging conjugation chemistry for antibodies as potential anti-cancer therapeutics

  • Bayan Alkhawaja

Student thesis: Doctoral ThesisPhD


This is a multidisciplinary research project in the field of bioconjugation chemistry. Bioconjugation chemistry, in its fundamental aspects, refers to the site specific covalent modification of bio-molecules adding or modulating desired characteristics. The recent advances in bioconjugation approaches have enabled the progressive construction of ground-breaking monoclonal antibodies (mAbs) biotherapies, such as antibody drug conjugates (ADCs) and bispecific antibodies. However, there is still a long list of limitations encountered by the currents approaches such as poor plasma stability, post-modification structural stability and heterogeneity of the produced conjugates, which are usually tackled by expensive and complex biological-based techniques. Therefore, in this study, we aimed to develop novel conjugation chemistry suitable for the construction of antibody-protein conjugates with promising anti-cancer activity.
Production of protein conjugates is usually achieved through targeting thiolate groups of their cysteine residues. We aimed to develop an effective reduction method for activation of thiolates towards conjugation reactions. We developed and evaluated a novel one-pot method by using water soluble azide-derivatised ethylene glycols (PEG-azides) to quench excess trialkylphosphines prior to thiol alkylation reactions. The rates of oxidation of trialkylphosphines with a series of PEG-azides were determined and the applicability of this in situ method was evaluated in conjugation reactions.
We have developed a novel rebridging approach of the reduced disulfide bonds of mAbs, based on an aryl bis-haloacetamide scaffold. The proposed scaffold was developed according to our findings related to the impact of a vicinal acetamide group on the reactivity and stability of various electrophiles. Interestingly, rebridging of mAbs with the bis-haloacetamide linkers has revealed a high efficiency in rebridging heavy-light and intra-heavy-heavy disulfide bonds affording half antibody (75 kDa) as a major rebridged product. Rebridging conditions were optimised and utilised to obtain rebridged bi-functional half antibody (75 kDa) modified with bio-orthogonal chemistry. The stability of the rebridged mAbs and the difference in reactivity and selectivity amongst bis-haloacetamide linkers with reduced mAbs have been thoroughly studied.
Moreover, we have developed an elegant bis-rebridging platform based on bis-haloacetamide scaffold. The bis-o-dihaloacetamide PEG linker has been applied in generating mAb conjugates with other proteins, including immunomodulating proteins and antibody fragments in an adaptable procedure with a very good yield. The promising findings imply the applicability of this approach to construct mAb immunoconjugates enabling multi-targets-based intervention in the oncology field.
Date of Award27 May 2019
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorJean Van Den Elsen (Supervisor) & Andrew Watts (Supervisor)

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