Antibody engineering studies using filamentous bacteriophage display technology.
thesisposted on 19.11.2015, 09:09 by Yi. Li
The work described here uses phage antibodies (PhAbs), combining site-directed mutagensis and molecular modelling to explore a number aspects of phage antibody engineering. In one study the characteristics of a phage-displayed multi-functional fusion protein were investigated. Bi-functional fusion proteins of staphylococcal IgG binding protein and a single chain Fv antibody (scFv) were displayed on the phage surface. Both moieties functioned although position in the fusion affected function. The protein A moiety of the displayed bi-functional protein provides an affinity handle to facilitate detection and purification of antibody fragments. The anti-hen egg lysozyme monoclonal antibody, HyHEL-10, has been a focus for studies antibody structure-function relationships. This antibody was used in the production and display on bacteriophage of a hybrid scFv which contained the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally-related but functionally distinct antibody, ASS 2. By using a combination of site-directed mutagenesis, complementary determining region (CDR) grafting and molecular modelling, a number of contact and non- contact residues that are important in determining the affinity of HyHEL-10 for lysozyme were identified. An important application of phage technology is in the creation of natural or synthetic antibody repertoires. A semisynthetic library was created by randomisation of heavy chain CDR3 of AS32 and antibody fragments with new specificities were selected from it. In a separate study using the "single pot" PhAb library (Nissim et al, 1994), to pan against intact germlings of the fungal plant parasite Phytophthora infestans and a peptide-BSA conjugate, two populations of antibodies were selected which recognised surface located epitopes of intact P. infestans and the peptide conjugate respectively. The surface location and distribution of the cognate epitopes of the anti-P. infestans PhAbs were confirmed by electron microscopy or fluorescence microscopy using rhodamine labelled PhAbs, illustrating the use of fluorescent PhAbs as immunolocalisation reagents.