Development of an inhibitory antibody fragment to human tissue factor using phage display technology

Abstract

Tissue factor is involved in the etiology of thrombotic diseases initiating the thrombosis associated with the inflammation that occurs during infection. The prevention of blood coagulation and inflammation is of primary importance in a number of pathological situations. A single-chain variable antibody fragment of molecular weight of 26 kD that inhibits the action of human tissue factor was selected by phage display technology, purified and tested for its tissue factor inhibitory effect, purified on a protein A column, and its purity evaluated on SDS-PAGE. The effects of the antibody fragment on prothrombin times, Factor Xa production, and thrombin generation were assessed with increasing fragment concentrations, using chromogenic and fluorometric substrates. The antibody fragment dose-dependently prolonged the prothrombin time (IC 50 5 0.5 mM) and delayed the lag phase before the thrombin generation burst and the peak thrombin concentration in the thrombin generation assay. The effect on thrombin generation was more pronounced in thrombophilic plasma than in normal plasma. Antibody-based tissue factor inhibitors therefore may provide an effective treatment for thrombotic disease without serious bleeding complications. Drug Dev Res 70 : 199-205, 2009.

The Use of Phage Display and Yeast Based Expression System for the Development of a Von Willebrand Factor Propeptide Assay: Development of a Von Willebrand Factor Propeptide Assay

“…The I library consists of diversified side chains on 18 residues while the J library has NNK side chains on these residues (N=GACT and K=GT). Antibody selection was done as previously described [ 8 ]. In short, Nunc-Immuno™ tubes (Thermo Scientific, USA) were coated with VWFpp displaying yeast cells (110 g/l in PBS) and rotated overnight at 4°C.…”