Download PDF
Semin Thromb Hemost 2007; 33(2): 185-195
DOI: 10.1055/s-2007-969033
Copyright © 2007 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA.

Single-Chain Antibodies as New Antithrombotic Drugs

Christoph E. Hagemeyer1 , Meike Schwarz2 , Karlheinz Peter1
  • 1Centre for Thrombosis and Myocardial Infarction, Baker Heart Research Institute, Melbourne, Australia
  • 2Department of Cardiology and Angiology, Albert-Ludwigs-University, Freiburg, Germany
Further Information

Publication History

Publication Date:
06 March 2007 (online)

    Permissions and Reprints

ABSTRACT

Antibodies are the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines. At present, several antibodies are approved for therapeutic use in diverse clinical settings, including oncology, chronic inflammatory diseases, transplantation, infectious diseases, and cardiovascular medicine. These approved antibody therapeutics include unmodified immunoglobulin G molecules, radioimmunoconjugates, antibody-drug conjugates, and fragment antigen-binding molecules. At least 150 additional antibodies are in clinical development. A major strength of therapeutic antibodies is their established properties as a drug class with high success rates from clinical trials to regulatory approvals. Much of the experience gained from the generation and optimization of one antibody is applicable to other antibodies. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as the smallest possible format for recombinant antibodies, and their use as antithrombotic drugs. We describe different antibody formats, the current applications of antibody fragments, and their generation by cloning from hybridoma cell lines as well as their selection from antibody libraries. We review the use of antibody fragments for thrombus targeting using fibrin and platelet-specific single-chain antibodies in combination with anticoagulants and thrombolytic agents as antithrombotic drugs.

KEYWORDS

Antibody fragments - hybridoma - thrombosis - phage display - glycoprotein (GP) IIb-IIIa

REFERENCES

  • 1 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.  Nature. 1975;  256 495-497
    PubMedSearch in Google Scholar
  • 2 Reichert J M, Rosensweig C J, Faden L B, Dewitz M C. Monoclonal antibody successes in the clinic.  Nat Biotechnol. 2005;  23 1073-1078
    PubMedSearch in Google Scholar
  • 3 Boulianne G L, Hozumi N, Shulman M J. Production of functional chimaeric mouse/human antibody.  Nature. 1984;  312 643-646
    PubMedSearch in Google Scholar
  • 4 Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy.  Nature. 1988;  332 323-327
    CrossrefPubMedSearch in Google Scholar
  • 5 Skerra A, Pluckthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli .  Science. 1988;  240 1038-1041
    CrossrefPubMedSearch in Google Scholar
  • 6 Toleikis L, Broders O, Dubel S. Cloning single-chain antibody fragments (scFv) from hybridoma cells.  Methods Mol Med. 2004;  94 447-458
    PubMedSearch in Google Scholar
  • 7 Kipriyanov S M, Moldenhauer G, Schuhmacher J et al.. Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics.  J Mol Biol. 1999;  293 41-56
    CrossrefPubMedSearch in Google Scholar
  • 8 Holliger P, Hudson P J. Engineered antibody fragments and the rise of single domains.  Nat Biotechnol. 2005;  23 1126-1136
    PubMedSearch in Google Scholar
  • 9 Chapman A P. PEGylated antibodies and antibody fragments for improved therapy: a review.  Adv Drug Deliv Rev. 2002;  54 531-545
    CrossrefPubMedSearch in Google Scholar
  • 10 Kipriyanov S M, Le Gall F. Generation and production of engineered antibodies.  Mol Biotechnol. 2004;  26 39-60
    CrossrefPubMedSearch in Google Scholar
  • 11 Kriangkum J, Xu B, Nagata L P, Fulton R E, Suresh M R. Bispecific and bifunctional single chain recombinant antibodies.  Biomol Eng. 2001;  18 31-40
    PubMedSearch in Google Scholar
  • 12 Hayden M S, Linsley P S, Gayle M A et al.. Single-chain mono- and bispecific antibody derivatives with novel biological properties and antitumour activity from a COS cell transient expression system.  Ther Immunol. 1994;  1 3-15
    PubMedSearch in Google Scholar
  • 13 Todorovska A, Roovers R C, Dolezal O, Kortt A A, Hoogenboom H R, Hudson P J. Design and application of diabodies, triabodies and tetrabodies for cancer targeting.  J Immunol Methods. 2001;  248 47-66
    CrossrefPubMedSearch in Google Scholar
  • 14 Simmons L C, Reilly D, Klimowski L et al.. Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies.  J Immunol Methods. 2002;  263 133-147
    CrossrefPubMedSearch in Google Scholar
  • 15 Carter P J. Potent antibody therapeutics by design.  Nat Rev Immunol. 2006;  6 343-357
    CrossrefPubMedSearch in Google Scholar
  • 16 Eshhar Z, Waks T, Bendavid A, Schindler D G. Functional expression of chimeric receptor genes in human T cells.  J Immunol Methods. 2001;  248 67-76
    CrossrefPubMedSearch in Google Scholar
  • 17 Chowdhury P S, Wu H. Tailor-made antibody therapeutics.  Methods. 2005;  36 11-24
    CrossrefPubMedSearch in Google Scholar
  • 18 Hoogenboom H R. Selecting and screening recombinant antibody libraries.  Nat Biotechnol. 2005;  23 1105-1116
    PubMedSearch in Google Scholar
  • 19 Coller B S. Anti-GPIIb/IIIa drugs: current strategies and future directions.  Thromb Haemost. 2001;  86 427-443
    PubMedSearch in Google Scholar
  • 20 Bhatt D L, Topol E J. Scientific and therapeutic advances in antiplatelet therapy.  Nat Rev Drug Discov. 2003;  2 15-28
    CrossrefPubMedSearch in Google Scholar
  • 21 Lonberg N. Human antibodies from transgenic animals.  Nat Biotechnol. 2005;  23 1117-1125
    PubMedSearch in Google Scholar
  • 22 Lonberg N, Taylor L D, Harding F A et al.. Antigen-specific human antibodies from mice comprising four distinct genetic modifications.  Nature. 1994;  368 856-859
    PubMedSearch in Google Scholar
  • 23 Jostock T, Dubel S. Screening of molecular repertoires by microbial surface display.  Comb Chem High Throughput Screen. 2005;  8 127-133
    CrossrefPubMedSearch in Google Scholar
  • 24 McCafferty J, Griffiths A D, Winter G, Chiswell D J. Phage antibodies: filamentous phage displaying antibody variable domains.  Nature. 1990;  348 552-554
    PubMedSearch in Google Scholar
  • 25 Kurtzman A L, Govindarajan S, Vahle K, Jones J T, Heinrichs V, Patten P A. Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins.  Curr Opin Biotechnol. 2001;  12 361-370
    CrossrefPubMedSearch in Google Scholar
  • 26 Jespers L, Schon O, Famm K, Winter G. Aggregation-resistant domain antibodies selected on phage by heat denaturation.  Nat Biotechnol. 2004;  22 1161-1165
    PubMedSearch in Google Scholar
  • 27 Hui K Y, Haber E, Matsueda G R. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen.  Science. 1983;  222 1129-1132
    PubMedSearch in Google Scholar
  • 28 Holvoet P, Laroche Y, Stassen J M et al.. Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody fused to single-chain urokinase.  Blood. 1993;  81 696-703
    PubMedSearch in Google Scholar
  • 29 Peter K, Graeber J, Kipriyanov S et al.. Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa.  Circulation. 2000;  101 1158-1164
    CrossrefPubMedSearch in Google Scholar
  • 30 Bode C, Hanson S R, Schmedtje Jr J F et al.. Antithrombotic potency of hirudin is increased in nonhuman primates by fibrin targeting.  Circulation. 1997;  95 800-804
    PubMedSearch in Google Scholar
  • 31 Weitz J I, Leslie B, Hudoba M. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors.  Circulation. 1998;  97 544-552
    PubMedSearch in Google Scholar
  • 32 Meyer B J, Badimon J J, Chesebro J H, Fallon J T, Fuster V, Badimon L. Dissolution of mural thrombus by specific thrombin inhibition with r-hirudin: comparison with heparin and aspirin.  Circulation. 1998;  97 681-685
    PubMedSearch in Google Scholar
  • 33 Loscalzo J. Thrombin inhibitors in fibrinolysis. A Hobson's choice of alternatives.  Circulation. 1996;  94 863-865
    PubMedSearch in Google Scholar
  • 34 Hagemeyer C E, Tomic I, Jaminet P et al.. Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide.  Thromb Haemost. 2004;  92 47-53
    PubMedSearch in Google Scholar
  • 35 Rand M D, Lock J B, van't Veer C, Gaffney D P, Mann K G. Blood clotting in minimally altered whole blood.  Blood. 1996;  88 3432-3445
    PubMedSearch in Google Scholar
  • 36 Abendschein D R, Baum P K, Verhallen P, Eisenberg P R, Sullivan M E, Light D R. A novel synthetic inhibitor of factor Xa decreases early reocclusion and improves 24-h patency after coronary fibrinolysis in dogs.  J Pharmacol Exp Ther. 2001;  296 567-572
    PubMedSearch in Google Scholar
  • 37 Zoldhelyi P, Bichler J, Owen W G et al.. Persistent thrombin generation in humans during specific thrombin inhibition with hirudin.  Circulation. 1994;  90 2671-2678
    PubMedSearch in Google Scholar
  • 38 Nicolini F A, Lee P, Malycky J L et al.. Selective inhibition of factor Xa during thrombolytic therapy markedly improves coronary artery patency in a canine model of coronary thrombosis.  Blood Coagul Fibrinolysis. 1996;  7 39-48
    PubMedSearch in Google Scholar
  • 39 Waxman L, Smith D E, Arcuri K E, Vlasuk G P. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa.  Science. 1990;  248 593-596
    CrossrefPubMedSearch in Google Scholar
  • 40 Sitko G R, Ramjit D R, Stabilito I I, Lehman D, Lynch J J, Vlasuk G P. Conjunctive enhancement of enzymatic thrombolysis and prevention of thrombotic reocclusion with the selective factor Xa inhibitor, tick anticoagulant peptide. Comparison to hirudin and heparin in a canine model of acute coronary artery thrombosis.  Circulation. 1992;  85 805-815
    PubMedSearch in Google Scholar
  • 41 Armstrong P W, Collen D, Antman E. Fibrinolysis for acute myocardial infarction: the future is here and now.  Circulation. 2003;  107 2533-2537
    CrossrefPubMedSearch in Google Scholar
  • 42 Dewerchin M, Vandamme A M, Holvoet P et al.. Thrombolytic and pharmacokinetic properties of a recombinant chimeric plasminogen activator consisting of a fibrin fragment D-dimer specific humanized monoclonal antibody and a truncated single-chain urokinase.  Thromb Haemost. 1992;  68 170-179
    PubMedSearch in Google Scholar
  • 43 Hagemeyer C E, Tomic I, Weirich U et al.. Construction and characterization of a recombinant plasminogen activator composed of an anti-fibrin single-chain antibody and low-molecular-weight urokinase.  J Thromb Haemost. 2004;  2 797-803
    CrossrefPubMedSearch in Google Scholar
  • 44 Liu Z H, Wan H Y, Wang B et al.. Construction and expression of a recombinant antibody-targeted plasminogen activator composed of a humanized monoclonal antibody against activated human platelets and a single-chain urokinase.  Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 1998;  30 601-606
    PubMedSearch in Google Scholar
  • 45 Bi Q, Cen X, Huang Y, Zhu S. Construction and characterization of trifunctional single-chain urokinase-type plasminogen activators.  Eur J Biochem. 2002;  269 1708-1713
    CrossrefPubMedSearch in Google Scholar
  • 46 Ding F X, Yan H L, Lu Y M et al.. Cloning, purification and biochemical characterization of a thrombus-ditargeting thrombolytic agent, comprised of annexin B1, ScuPA-32K and fibrin-adherent peptide.  J Biotechnol. 2006;  126 394-405
    CrossrefPubMedSearch in Google Scholar
  • 47 Ding B S, Gottstein C, Grunow A et al.. Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature.  Blood. 2005;  106 4191-4198
    CrossrefPubMedSearch in Google Scholar
  • 48 Dasgupta H, Blankenship J C, Wood G C, Frey C M, Demko S L, Menapace F J. Thrombocytopenia complicating treatment with intravenous glycoprotein IIb/IIIa receptor inhibitors: a pooled analysis.  Am Heart J. 2000;  140 206-211
    CrossrefPubMedSearch in Google Scholar
  • 49 Peter K, Schwarz M, Ylanne J et al.. Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (alphaIIbbeta3) inhibitors.  Blood. 1998;  92 3240-3249
    PubMedSearch in Google Scholar
  • 50 Quinn M J, Byzova T V, Qin J, Topol E J, Plow E F. Integrin alphaIIbbeta3 and its antagonism.  Arterioscler Thromb Vasc Biol. 2003;  23 945-952
    PubMedSearch in Google Scholar
  • 51 Schwarz M, Rottgen P, Takada Y et al.. Single-chain antibodies for the conformation-specific blockade of activated platelet integrin alphaIIbbeta3 designed by subtractive selection from naive human phage libraries.  FASEB J. 2004;  18 1704-1706
    PubMedSearch in Google Scholar
  • 52 Billheimer J T, Dicker I B, Wynn R et al.. Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated.  Blood. 2002;  99 3540-3546
    CrossrefPubMedSearch in Google Scholar
  • 53 Schwarz M, Meade G, Stoll P et al.. Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets.  Circ Res. 2006;  99 25-33
    CrossrefPubMedSearch in Google Scholar

Karlheinz PeterM.D. 

Baker Heart Research Institute

P.O. Box 6492 St Kilda Road Central, Melbourne, Victoria 8008, Australia

Email: karlheinz.peter@baker.edu.au