Expression and interaction studies of recombinant human monoclonal antibodies

Abstract: The highly specific binding characteristics of monoclonal antibodies (mAbs) have made them one of the most useful tools in the fields of research and biotechnology where they provide the basis for a large number of diagnostic and technical assays. In recent years, with the advent of techniques to generate fully human antibodies, mAbs have also emerged as a new important class of therapeutics. Accordingly, improved technologies for faster isolation and characterization of mAbs are of great demand. The studies included in this thesis are all related to monoclonal antibody technologies. In order for a new mAb to be characterized, it has to be produced in large quantities. This has been problematic since established production systems for recombinant antibodies either express at too low levels, are time consuming or are unreliable only allowing successful expression of certain clones. We explored the Drosophila S2 cell line for high-level expression of mAbs in the form of human IgG1. A series of vectors were designed to allow easy transfer of isolated mAb genes from hybridomas and commonly used phage-display vectors. The S2 cells showed a number of advantages including rapid establishment of stable cellines that express at high levels, easy culturing and easy production scale-up. The S2 produced mAbs had indistinguishable binding properties compared to mAbs produced in mammalian CHO cells as shown by antigen binding in ELISA, immunofluorescent staining of cells and virus neutralization capacity. Next, we used the S2 system to produce three previously isolated mAbs targeting the envelope E2 of the highly sequence variable Hepatitis C virus (HCV). The subsequent characterization of the mAbs showed that two of them bound and neutralized a broad range of HCV isolates. The epitopes of both mAbs were mapped to a highly conserved region of E2 strongly indicated to be involved in interaction with CD81, a cell receptor important for HCV cell entry. In a following study, a new mAb targeting the adhesion protein BabA of the human pathogen Helicobacter pylori was isolated from an immune combinatorial scFv antibody library. BabA is involved in H. pylori adherence to fucosylated blood sugars such as Lewis b (Leb) on the gastric mucosa. Again, the S2 system was used for high level production of mAb in IgG1 format allowing a detailed characterization of the antibody specificity profile. The mAb bound BabA derived from a wide range of H pylori strains. Finally, we investigated the possibilities to develop a system for high throughput screening of mAb-antigen interactions in living cells. Such a system would be of great use since the antigen is displayed in its natural environment. This allows screening of antibodies targeting antigens not suitable for protein purification such as membrane proteins. Our system was based on Förster Resonance Energy Transfer (FRET). To circumvent background signal problems associated with established FRET-pairs, we investigated a novel pair of fluorescent proteins and subsequently showed its potential in library screenings by FACS sorting. However, proteins bound to the ER-membrane were suboptimal for analysis in this system, as false-positive FRET signals were observed when investigating interactions between antigen-antibody anchored to the ER.