SELECTION AND APPLICATION OF HUMAN SINGLE-CHAIN FV ANTIBODY FRAGMENTSFROM A SEMISYNTHETIC PHAGE ANTIBODY DISPLAY LIBRARY WITH DESIGNED CDR3 REGIONS

We have constructed a large (3.6x10(8) clones) phage display library o f human single chain Fv (scFv) antibody fragments by combining 49 germ line V-H genes with synthetic heavy chain CDR3 (HCDR3) regions and sev en light chains. The HCDR3 regions varied in length between 6 and 15 r esidues and were designed to contain fully randomized stretches of ami no acid residues flanked by regions of limited residue variability tha t were composed of amino acid residues that frequently occur in natura l antibodies. We reasoned that this approach would increase the freque ncy of functional molecules in our library and, in addition, permit us to efficiently utilize available cloning space. By direct selection o n solid phase-bound antigens we obtained phage antibodies with binding activities to 13 different antigens, including Von Willebrand factor, the DNA-binding HMG box of transcription factor TCF-1 and the tumor a ntigen EGP-2. In addition, we applied a competitive selection procedur e to target phage antibodies to the desired portion of a recombinant f usion protein and to select phage antibodies capable of discriminating between the two highly homologous homeobox proteins PBX1a and PBX2. T he functional capacity of monoclonal phage antibodies was assessed in immuno-histochemical staining of tissue specimens, Western blotting as says and immunofluorescent analysis of cells by flow cytometry The res ults demonstrate that this large human phage antibody library contains a broad assortment of binding specificities that can be applied in a variety of biochemical assays.