Structural stability and binding properties of soluble and membrane-anchored recombinant antibodies

Antibody engineering and advances in microbial expression systems have enabled production of small, active antibody fragments. However, the functional expression yields of these recombinant antibodies vary widely. The results described in this thesis elucidate factors influencing the expression of stable and active antibody fragments in bacteria. The model antibody used throughout this study was a mouse monoclonal antibody binding to 2-phenyloxazolone (Ox). It was shown that the first constant domain of the heavy chain (CH1) has a remarkable effect on secretion of functional and stable Fab fragments. Comparison of the production of the Ox IgG1 and Ox IgG3 subclass Fab fragments in bacteria demonstrated the superiority of the Ox IgG1 Fab compared to the Ox IgG3 Fab. In addition to its effect on secretion, the CH1 domain contributes to the thermal stability of the antibody fragments. To study the effect of a linker peptide on both proteolytic stability and binding activity of singlechain antibodies, six different Ox scFv derivatives and an Ox Fv fragment with no joining peptide between the variable domains were constructed. It was shown that joining of the variable domains with a linker peptide improved hapten binding properties compared to the Ox Fv fragment, but may expose the fragment to proteolytic degradation. Truncation of the linker peptide to less than 12 amino acids induced formation of dimers or multimers. The binding affinities determined for the monomeric Ox scFv and Ox IgG1 Fab fragments using the BIAcore biosensor and fluorescence quenching methods were close to each other and comparable to that of the parental monoclonal antibody. In addition to studies with soluble antibody fragments, this work was extended to cover characterization of antibody fragments displayed on liposomes and on the surface of baculovirus. Immunoliposomes have potential applications both in therapy and in immunodiagnostics. In this work liposomes displaying antibodies were generated by incorporation of purified lipid-tagged Ox scFvs expressed in