New strategies for cysteine bioconjugation and protein cross-linking

• Main Author: Forte, Nafsika
• Other Authors: Baker, James
• Published: University College London (University of London) 2018
• Subjects: 540
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.756269

The connection of two or more proteins with different binding specificities or activities can lead to unique and powerful systems with significant potential in therapeutic applications, particularly oncology. While a plethora of active bifunctional proteins have been produced by fusion, this approach is not always viable, since modification of the N- or C- terminus can compromise the proteins’ bioactivities, with complications during folding and processing often being observed. Chemical cross-linking can offer a more versatile strategy towards the synthesis of protein-protein conjugates. However, current state-of-the-art approaches are either not selective, yielding heterogeneous mixtures or require mutations with natural or unnatural amino acids, which are often associated with low expression yields and limited applicability towards all protein systems. Recently, a bis-dibromomaleimide cross-linking reagent was used to create a bispecific scFv-Fab antibody in a site-specific manner, by targeting and bridging disulfide bonds. In this work, the application of these cross-linking reagents was extended into the conjugation of proteins containing different reactive handles, as well as sterically hindered conjugation sites. Diiodo-maleimides were shown to offer the ideal properties of rapid bioconjugation and importantly, reduced hydrolysis, which was identified as a significant factor affecting the conjugation efficiency. The optimized linkers were exploited to link human serum albumin to antibody fragments (scFv and Fab) as a prospective half-life extension platform, with retention of antigen binding and robust serum stability. The linker design was also extended to incorporate a third point of attachment for another biomolecule. An anti-CEA scFv homotrimer was formed, by disulfide bridging, which was shown to infer a combination of greater antigen avidity and increased in vivo half-life. Finally, with the view to developing a Bispecific T-cell Engager (BiTE) construct that could lead to more efficient cell-killing, an anti-CEA/ anti-CD3 scFv heterotrimer of increased avidity towards CD3 antigen was developed, showing the potential of this strategy for the construction of a diverse range of bioconjugates. The last part of this thesis describes investigations towards the development of a new methodology that selectively modifies specific lysine residues, via acyl transfer from the disulfide bond of Fab antibody fragment. This approach is expected to complement disulfide bridging as a strategy for selective modification, but also be advantageous for systems where this could result in detrimental changes to the antibody structure, offering a highly promising platform for bioconjugation.