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(Circulation. 2000;101:1158.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa

Presented in part at the annual meeting of the American College of Cardiology, New Orleans, La, March 7–10, 1999, and published in abstract form (J Am Coll Cardiol. 1999;33:2A).

Karlheinz Peter, MD; Justin Graeber, BS; Sergey Kipriyanov, PhD; Monika Zewe-Welschof, PhD; Marschall S. Runge, MD, PhD; Wolfgang Kübler, MD; Melvyn Little, PhD; Christoph Bode, MD

From the Department of Cardiology, University of Freiburg (K.P., C.B.), Freiburg, Germany; Department of Cardiology, University of Heidelberg (J.G., W.K.), Heidelberg, Germany; Sealy Center for Molecular Cardiology, University of Texas (M.S.R.), Galveston, Tex; and the German Cancer Research Center (S.K., M.Z.-W., M.L.), Heidelberg, Germany.

Correspondence to Dr Karlheinz Peter, Internal Medicine III, University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany. E-mail peter{at}mm31.ukl.uni-freiburg.de

Background—Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot.

Methods and Results—The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly₄Ser)₃ was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the Bß15-22 peptide of fibrin (ß-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv_59D8 and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to ß-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin.

Conclusions—A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.

Key Words: anticoagulants • antibodies • thrombosis • molecular biology

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