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 blocks tissue factor function even in the presence of bound factor VIIa. The cloned murine D3 antibody was humanized and affinity matured by exchanging amino acids in the complementarity determining regions as well as in the antibody framework. The humanized antibody, D3H44, bound to tissue factor with a 100-fold increased affinity (KD 0.1 nM) as compared to the original murine and chimeric versions. Depending on the particular disease, different pharmacokinetic properties of the antibody may be required and, therefore, several antibody variants-- F(ab), F(ab')2, IgG2, IgG4 and IgG4b-were generated. In vitro, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor: factor VIIa-mediated activation of factors IX and X (e.g. D3H44-F(ab')2, IC50(F.X) 47 pM). In addition, D3H44-F(ab')2 completely prevented fibrin deposition in a human ex vivo thrombosis model under venous blood flow conditions (IC50 37 nM). The humanized D3 antibodies may be utilized for treatment of cardiovascular diseases which involve tissue factor activity, e.g. acute coronary syndrome and venous thrombosis.
Presta
L
L
Department of Immunology, Genetech Inc, South San Francisco, CA 94080, USA. Presta.leonard@gene.com
Sims
P
P
Meng
Y G
YG
Moran
P
P
Bullens
S
S
Bunting
S
S
Schoenfeld
J
J
Lowe
D
D
Lai
J
J
Rancatore
P
P
Iverson
M
M
Lim
A
A
Chisholm
V
V
Kelley
R F
RF
Riederer
M
M
Kirchhofer
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use*; D000915:Antibody Affinity / Q000276:immunology*; D001780:Blood Coagulation Tests; D003001:Cloning, Molecular; D005164:Factor IX / Q000187:drug effects / Q000378:metabolism; D005170:Factor X / Q000187:drug effects / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D016415:Sequence Alignment; D013925:Thromboplastin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D005164:Factor IX; D005170:Factor X; D013925:Thromboplastin