Generation of a humanized,high affinity anti-tissue factor antibody for use as a novel antithrombotic therapeutic

Blocking the cofactor function of human tissue factor may be beneficial in various coagulation-mediated diseases. The murine antibody D3 binds to the membrane proximal substrate interaction region of human tissue factor and b locks tissue factor function even in the presence of bound facto; Wa. The c loned murine D3 antibody was humanized and affinity matured by exchanging a mino acids in the complementarity determining regions as well as in the ant ibody framework. The humanized antibody, D3H44, bound to tissue factor with a 100-fold increased affinity (K-D 0.1 nM) as compared to the original mur ine and chimeric versions. Depending on the particular disease, different p harmacokinetic properties of the antibody may be required and, therefore, s everal antibody variants - F(ab), F(ab')(2), IgG2, IgG4 and IgG4b - were ge nerated. In vitro, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor: factor Wa-mediated activation of fact ors IX and X (e.g. D3H44-F(ab')(2), IC50 ((F.X)), 47 pM). In addition, D3H4 4-F(ab')(2) completely prevented fibrin deposition in a human ex vivo throm bosis model under venous blood flow conditions (IC50 37 nM). The humanized D3 antibodies may be utilized for treatment of cardiovascular diseases whic h involve tissue factor activity, e. g. acute coronary syndrome and venous thrombosis.