Generation of a Humanized, High Affinity Anti-tissue Factor Antibody for Use as a Novel Antithrombotic Therapeutic

Leonard Presta; Paul Sims; Y. Gloria Meng; Paul Moran; Sherron Bullens; Stuart Bunting; Jill Schoenfeld; David Lowe; Jadine Lai; Patricia Rancatore; Mark Iverson; Amy Lim; Vanessa Chisholm; Robert F. Kelley; Markus Riederer; Daniel Kirchhofer

doi:10.1055/s-0037-1615593

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 85(03): 379-389
DOI: 10.1055/s-0037-1615593

Rapid Communications

Schattauer GmbH

Generation of a Humanized, High Affinity Anti-tissue Factor Antibody for Use as a Novel Antithrombotic Therapeutic

Leonard Presta

,

Paul Sims

²BioAnalytical Technology

,

Y. Gloria Meng

²BioAnalytical Technology

,

Paul Moran

³Cardiovascular Research

,

Sherron Bullens

³Cardiovascular Research

,

Stuart Bunting

³Cardiovascular Research

,

Jill Schoenfeld

³Cardiovascular Research

,

David Lowe

³Cardiovascular Research

,

Jadine Lai

,

Patricia Rancatore

⁴Recovery Sciences

,

Mark Iverson

⁴Recovery Sciences

,

Amy Lim

⁴Recovery Sciences

,

Vanessa Chisholm

⁵Molecular Biology

,

Robert F. Kelley

⁶Protein Engineering, Genentech Inc., South San Francisco, CA, USA

,

Markus Riederer

⁷Preclinical Research Department, F. Hoffmann-La Roche Ltd., Basel, Switzerland

,

Daniel Kirchhofer

³Cardiovascular Research

› Author Affiliations

Abstract

Summary

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 (K_D 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.

Keywords

Tissue factor - coagulation factor VII - humanization - anticoagulants - thrombosis

Full Text

References

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