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Title Δημιουργία Βιβλιοθηκών έκφρασης αντισωμάτων σε φάγους και απομόνωση ανθρωπίνων ANTI-MUC1 ανασυνδυασμένων αντισωμάτων
Creator Kandilogiannaki, Maria
Abstract The aim of the thesis was the isolation and characterization of human anti-MUC1 recombinant antibodies. MUC1 belongs to a family of high molecular weight glycoproteins (mucins), which are heavily glycosylated and are expressed by normal and malignant tissues. The tumor-associated antigen MUC1 is overexpressed and underglycosylated in human adenocarcinomas of diverse origin, such as breast, ovary and colon. The technology of phage display was used as a tool for the isolation of specific anti-MUC1 antibodies. We constructed scFv and Fab antibody libraries by isolating RNA from human B cells which were immunized in vitro with the MUC1 and MUC2 VNTR peptide epitopes. The antibody libraries were selected with synthetic biotinylated MUC1 100mer peptide corresponding to five tandem repeats of the protein core and MUC2 46mer peptide corresponding to two tandem repeats of the protein core. The antibody molecules which were isolated, didn't present a satisfactory anti-MUC1 or anti-MUC2 specificity. Two other libraries were selected with the synthetic MUC1 peptide: one naive scFv antibody library and one immunized Fab antibody library. In the first case, five human anti-MUC1 scFv antibodies were isolated. Two of them were highly specific for MUC1 as was analysed by ELISA, flow cytometry, BIAcore and immunohistochemistry. The best scFv molecule, 10A, appeared to distinguish normal cells from adenocarcinoma cells, which makes it an important candidate for use in antibody-based tumor targeting. Finaly we investigated whether four E. coli. strains that were deficient in the periplasmic proteases tsp, protease III, degP, ompT, in different combinations, affect the expression levels of the best anti-MUC1 scFv antibody fragment, 10A. The ompT protease appeared to be involved in partial degradation of the scFv since degradation products were observed in all ompT unmutated strains in Western blotting, whereas such products were absent in the ompT mutated strains. The HM120 strain that contained most mutations, expressed the antibody fragment efficiently but the level of functional antibody activity was low. This was probable due to an accumulation of incorrectly folded antibody molecules in the periplasm as it was characterised by low enzyme immunoassay reactions in contrust to the intense staining of the tag in Western blots. The better understanding of the periplasmic protease involvement in the process of the antibody expression in bacteria may allow us to design host E. coli strains that are more efficient in producing functional antibodies.
Issue date 2001-01-01
Date available 2001-02-05
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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