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

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 single-chain 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 E. coli into phospholipid liposomes.It was demonstrated that the biosynthetically lipid-tagged Ox scFv molecules can be immobilized in a functional, stable and oriented manner onto liposomal membranes, resulting in immunoliposomes showing specific hapten binding.BIAcore analysis of the immunoliposomes revealed very slow dissociation from the Ox surface, which is in good agreement with the predicted multivalent nature of the immunoliposomes.The baculoviral display approach holds a promise as a candidate for a eukaryotic display system.In this work single-chain antibodies were used as a model to investigate functional expression of foreign proteins on the surface of baculovirus.Viral vectors displaying cell targeting moieties such as single-chain antibodies have aroused interest as gene transfer vehicles.Two Ox scFv derivatives and a human scFv specific for carcinoembryonic antigen (CEA) were fused to the major envelope protein (gp64) of the Autographa californica nuclear polyhedrosis virus (AcNPV).It was shown that the two single-chain antibodies which contained a (GGGGS)3 linker peptide were incorporated into the budded virus particles and expressed as functional hapten/antigen binding fragments on the viral surface.In the case of the third scFv containing a natural linker peptide derived from a fungal cellulase, less efficient incorporation into the virus particles was observed.