Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa.
BACKGROUND
Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot.


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 (Gly(4)Ser)(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 Bbeta15-22 peptide of fibrin (beta-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 beta-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.
Peter
K
K
Department of Cardiology, University of Freiburg, Freiburg, Germany. peter@mm31.ukl.uni-freiberg.de
Graeber
J
J
Kipriyanov
S
S
Zewe-Welschof
M
M
Runge
M S
MS
Kübler
W
W
Little
M
M
Bode
C
C
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000991:Antithrombins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D004926:Escherichia coli; D005069:Evaluation Studies as Topic; D015951:Factor Xa / Q000378:metabolism*; D005337:Fibrin / Q000276:immunology*; D005822:Genetic Vectors; D006629:Hirudins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D000991:Antithrombins; D006629:Hirudins; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D015951:Factor Xa