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

We have constructed a large (3.6x10(8) clones) phage display library of human single chain Fv (scFv) antibody fragments by combining 49 germline V-H genes with synthetic heavy chain CDR3 (HCDR3) regions and seven light chains. The HCDR3 regions varied in length between 6 and 15 residues and were designed to contain fully randomized stretches of amino acid residues flanked by regions of limited residue variability that were composed of amino acid residues that frequently occur in natural antibodies. We reasoned that this approach would increase the frequency of functional molecules in our library and, in addition, permit us to efficiently utilize available cloning space. By direct selection on 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 antigen EGP-2. In addition, we applied a competitive selection procedure to target phage antibodies to the desired portion of a recombinant fusion protein and to select phage antibodies capable of discriminating between the two highly homologous homeobox proteins PBX1a and PBX2. The functional capacity of monoclonal phage antibodies was assessed in immuno-histochemical staining of tissue specimens, Western blotting assays and immunofluorescent analysis of cells by flow cytometry The results 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.