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, VH-(Gly4Ser)3-VL) 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.
Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate / S. Debrock, L. Sironi, P.J. Declerck. - In: GENE. - ISSN 0378-1119. - 189:1(1997 Apr 11), pp. 83-88. ((Intervento presentato al 10. convegno Forum for Applied Biotechnology tenutosi a Brugge nel 1996.
|Titolo:||Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate|
|Parole Chiave:||serpin; PAI-1; monoclonal antibody; single-chain variable fragment|
|Settore Scientifico Disciplinare:||Settore BIO/14 - Farmacologia|
|Data di pubblicazione:||11-apr-1997|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/S0378-1119(96)00838-4|
|Appare nelle tipologie:||01 - Articolo su periodico|