Site-specific chemical modification of antibodies for the modulation of function

Thesis

Chemical modification of antibodies is critical for many research areas including therapeutic and biotechnological applications. In particular, strategies for site-specific chemical modification via non-natural amino acids forming homogenous immunoconjugates are of interest. The use of fully functional single-domain antibodies derived from naturally occurring heavy chain antibodies in Camelidae species is attractive due to their enhanced properties, which are discussed in this piece of work. In terms of chemical antibody modification, much of the existing research is focused on modification away from binding regions, thus minimising disturbance to antibody function. In this thesis however, modifications within the binding region of the single-domain antibody cAb-Lys3 are described in a site-specific fashion with the aim of modulating binding affinities. An efficient and high yielding method for the expression and purification of cAb-Lys3 is described, followed by the site-specific installation of dehydroalanine, an electrophilic non-natural amino acid, able to react with nucleophiles that are inert to reaction with other proteinogenic amino acids. Then, the use of dehydroalanine as a unique reaction handle is explored. Firstly, the addition of short alkyl chains to dehydroalanine within the binding region of cAb-Lys3 is described with the aim of increasing hydrophobic interactions when binding to antigen. This has been attempted in two ways – via the addition of thiols, and by zinc-mediated radical additions. Secondly, the development of an antibody-based AND logic gate, converting cAb-Lys3 to a multi input protein requiring more than antigen for its binding is described. Site-specific chemical methods via dehydroalanine are utilised, and the application of the antibody-based AND logic gate in vitro and in mammalian tissue are discussed, as well as the prospects of the research.

Authors: Gunnoo, S; S. B. Gunnoo

• Publication date: 2013
• DOI: 10.5287/ora-5ro6wawbv
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: logic gate; site-specific protein modification; cAb-Lys3; dehydroalanine; protein modification; post-translational modification mimic; site-selective; single-domain antibodies; antibody
• Subjects: Protein chemistry; Organic chemistry; Chemical Biology