Bactericidal activity and improvement of therapeutic potency of recombinant antibody fragments
|Hall, J. Christopher
|Department of Environmental Biology
|University of Guelph
|Doctor of Philosophy
|In addition to mediating the elimination of pathogens via phagocytosis and immune effector cell- or complement-dependent cytotoxicity, certain antibodies exert antimicrobial functions through catalytic activities toward their target antigens or interference with the biological functions of target cells. Here, an anti-'Pseudomonas aeruginosa' 06ad lipopolysaccharide (LPS) IgG1 is reported to be capable of killing its target bacteria independent of immune effector cells and complement. Therefore, the first objective of this thesis was to investigate the bactericidal activity and mechanism of the anti-'P. aeruginosa' O6ad IgG1. This antibody and its Fab (fragment antigen binding) and scFv (single-chain variable fragment) fragments displayed remarkable bactericidal activities specifically against ' P. aeruginosa' O6ad in dose- and time-dependent manners 'in vitro', and provided 'in vivo' protection against ' P. aeruginosa' O6ad infection in a leukopenic mouse model. These molecules executed bactericidal effects by disrupting the bacterial cell wall as well as by inhibiting cell division via a mechanism involving antigen binding. The bactericidal function of these molecules indicates the potential for developing novel therapeutic applications. Recombinant antibody fragments (rAbF) are promising reagents in diagnostics and therapeutics. However, their therapeutic application is often compromised by their short serum half-lives and their inability to recruit host immune effectors. Therefore, the second objective of this thesis was to improve the therapeutic potency of rAbFs by non-covalent linking an epitope-tagged rAbF with an anti-epitope tag monoclonal antibody (mAb), resulting in the formation of a bivalent rAbF-mAb complex. This study showed that following complex formation with an anti-tag IgG (i.e., anti-Penta-His or anti-c-myc IgGs), the therapeutic potencies of both anti-'P. aeruginosa' Fab and scFv, tagged with c-myc and 6xHis, were significantly improved in their antigen-binding capacities, pharmacokinetic profiles, and their ability to mediate fragment crystallizable (Fc) region-dependent effector functions (i.e., complement deposition and bacterial phagocytosis). These results demonstrate that the complex formation with a full-length mAb is a simple and effective strategy for improving the therapeutic potential of rAbFs.
|University of Guelph
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|recombinant antibody fragments
|Bactericidal activity and improvement of therapeutic potency of recombinant antibody fragments