Development of protein VII and protein IX as the new platforms for phage display.

摘要

This thesis comprises efforts toward the development of the minor coat proteins pVII and pIX of filamentous bacteriophage as new platforms for phage display, as well as the construction and investigation of a pIII-displayed scFv library. The research is described in four chapters.; In chapter one, the orientations of pVII and pIX were verified and demonstrated to be suitable for the display of a heterodimeric array using the antibody structure as a classic paradigm. The well-studied catalytic antibody PCP21H3 was used as an example and its variable regions of heavy chain and light chain were fused to the N-termini of pVII and pIX, respectively. Significantly, the phage-displayed Fv maintained fully functional binding and catalytic activities.; In chapters two and chapter three, the pVII and pIX technology was taken a step further so as to construct a novel random peptide library and a naive human scFv library. The peptide library was panned against B-lymphocyte WI-L2 cells with the aim of isolating internalizing peptides. After five rounds of panning, one unique peptide-phage, designated CHL8, was identified that specifically bound to and penetrated the cells. The quality and utility of a naive human scFv library of 4.5 x 109 members displayed on pIX was examined by panning against six different protein antigens. Specific antibodies against all six antigens were obtained and more than 90% of the clones showed positive binding for their respective antigens after as few as three rounds of panning. Furthermore, antibodies with nanomolar to subnanomolar affinity were directly isolated from this naive human scFv library against antigens such as SEB and CTB.; In chapter four, a pIII phage-displayed naive human scFv library was harnessed to isolate tumor-specific and internalizing antibodies by direct selection against tumor cell lines together with normal cell line subtraction. Three scFvs, SW1, PAN10, and PR5 were isolated from panning and their receptors were identified to be human integrin alpha3beta1 (SW1, PAN10) and the transferrin receptor (PR5). The scFv SW1 was studied in further detail and found to induce functional effects as a ligand-mimetic by mediating cell adhesion and migration and the functional activities were integrin-alpha3 dependent.