Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin

Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires, However, the affinity of t he selected antibodies is usually low and current methods of affinity matur ation are complex and time-consuming. In this paper, we describe an easy wa y to increase the functional affinity (avidity) of single chain variable fr agments (scFvs) by tetramerization on streptavidin, following their site-sp ecific biotinylation by the enzyme BirA. Expression vectors have been const ructed that enable addition of the 15 amino acid biotin acceptor domain (BA D) on selected scFvs. Different domains were cloned at the C-terminus of sc Fv in the following order: a semi-rigid hinge region (of 16 residues), tile BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-imm une and murine immune phage display libraries. The scFvs were first synthes ized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatogra phy. Purified biotinylated scFvs were tetramerized on the streptavidin mole cule to create a streptabody (StAb). Tile avidity of various forms of anti- CEA StAbs. tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the sc Fv monomer counterparts. Furthermore, the percentage of direct binding of I -125-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expre ssing cells showed a dramatic increase For the tetramerized scFv (> 80%), a s compared with the monomeric scFv (< 20%). Interestingly, the percentage b inding of I-125-labeled anti-CEA StAbs to CEA-expressing colon carcinoma ce lls was definitely higher (> 80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a S tAb format was demonstrated by Western blot analysis, where tetramerized an ti-CEA scFv could detect a small quantity of CEA at a concentration 100-fol d lower than the monomeric scFv. (C) 2001 Elsevier Science Ltd. All rights reserved.