Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate

Sophie Debrock, Luigi Sironi, Paul J. Declerck

Research output: Contribution to journalArticlepeer-review

Abstract

Increased levels of plasminogen activator inhibitor-1 (PAI-1) are a well-known risk for cardiovascular diseases. A significant number of investigations are aimed at lowering plasma levels of PAI-1 to enhance endogenous fibrinolysis. We have recently generated monoclonal antibodies that neutralize PAI-1 activity by switching the inhibitory conformation to a substrate conformation. However, intact murine antibodies have quite some disadvantages for therapeutic use in man. In the current study, we describe the construction of a smaller antibody fragment derived from a monoclonal antibody (MA-8H9D4) with PAI-1 neutralizing properties. The cDNAs encoding the variable domains of the heavy and light chain were amplified, linked and cloned into a phagemid vector. Resulting clones were expressed as a single-chain variable fragment (scFv, V(H)-(Gly4Ser)3-V(L)) on the surface of a phage and selected for binding to PAI-1. Subsequently, a positive phage was used for the production of soluble scFv-8H9D4. Following purification, the characteristics of the scFv-8H9D4 were compared to those of the original MA-8H9D4. The scFv inhibited PAI-1 activity to a similar extent as MA-8H9D4 and by a similar mechanism, i.e., induction of a conformational switch. Thus, this smaller antibody fragment, exhibiting the same properties as the parent molecule may constitute a useful starting point for the design of PAI-1 neutralizing therapeutics.

Original languageEnglish
Pages (from-to)83-88
Number of pages6
JournalGene
Volume189
Issue number1
DOIs
Publication statusPublished - Apr 11 1997

Keywords

  • Monoclonal antibody
  • PAI-1
  • Serpin
  • Single-chain variable fragment

ASJC Scopus subject areas

  • Genetics

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