The development of disulphide-bridging chemistry for the generation of antibody-protein conjugates

  • Beccie Martin

Student thesis: Doctoral ThesisPhD

Abstract

Bioconjugation techniques have been used to produce pioneering antibody-based therapeutics including antibody-drug conjugates (ADCs) and bispecific antibodies (BsAbs), which are revolutionising the treatment of many cancers. Bioconjugation refers to the selective modification of existing biomolecules with novel and desired functions. It is also a key principle in many diagnostic and research tools including fluorescent imaging and the highly sensitive detection technique the enzyme-linked immunosorbent assay (ELISA).
Classical methods of antibody conjugation often result in heterogenous bioconjugates which suffer from reduced thermal stability and therapeutic potency. To achieve a more site- selective conjugation, highly complex and expensive modification to the antibody structure is often employed. Our aim during this research was to develop a method of selective chemical conjugation to native antibodies to be used in the construction of homogenous antibody conjugates.
Disulphide-bridging reagents are a new class of conjugation chemistry that can attach across a reduced antibody disulphide bond to lower the number of conjugation sites on an antibody. A selection of bis-haloacetamide linkers were evaluated as disulphide-bridging reagents. Through optimisation of antibody reduction and conjugation conditions highly selective re- bridging of the interchain disulphide bond between the heavy and light chain was achieved. Full bridging of the disulphide bonds resulted in disulphide scrambling at the hinge region. Using biolayer interferometry, we found disulphide scrambling did not significantly affect trastuzumab binding to the FcgRIIA and FcgRIIIA receptors.
We also demonstrate the suitability of a bis-1,2-diiodoacetamide PEG7 homobifunctional crosslinker with disulphide-bridging terminals for crosslinking Fabs and Mabs. Bispecific antibodies were conveniently generated using this crosslinker in a range of formats in good yield. Bispecific antibody generation was also approached from a click-chemistry perspective. Disulphide-bridging linkers with DBCO and azide handles were synthesized and used in SPAAC reactions to afford fluorescently-labelled antibodies and bispecific antibodies. Both crosslinking techniques show great potential for constructing homogenous antibody conjugates with potent anti-cancer activity.
Date of Award16 Nov 2022
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorAndrew Watts (Supervisor) & Jean Van Den Elsen (Supervisor)

Keywords

  • Disulphide bridging
  • Bioconjugation
  • Haloacetamide
  • Antibodies
  • Re-bridging

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