Molecular cloning and characterization of an antibody fragment (SCFV) that converts plasminogen activator inhibitor-1 from inhibitor to substrate

Plasminogen activator inhibitor-1 (PAI-1) can occur in three different interconvertible conformations: an active, a latent and a substrate form. We have previously characterized monoclonal antibodies (MAs) with neutralizing properties towards PAI-1. However, these antibodies (Mr=150 kDa) are unlikely to be succcsful for use as therapeutic agents with PAI-1 modulating properties. In this study we report the cloning of the variable domains of the heavy and light chain of one of these antibodies (MA-8H9D4), the expression (in E.coli) as a single-chain variable fragment (scFv) and the characterization of its functional properties. The percentage inhibition of the PAI-1 activity by scFv-8H9D4 (5-fold molar excess) was similar to that observed with the intact antibody (16-fold molar excess) (97 ±16 % and 88 ±10 %, respectively, mean SD, n=3). Furthermore, their influence on the reaction products formed during interaction of PAI-1 with tissue-type plasminogen activator (t-PA), as evaluated by SDS-PAGE followed by densitometric scanning, was also comparable. control MA-8H9D4 scFv-8H9D4 active PAI-1 51±11* 14±5 15±5 latent PAI-1 40±10 45±3 37±12 substrate PAI-1 9±2 42±3 45±4 * expressed as % of total PAI-1 antigen; mean ± SD (n=3); MA-8H9D4 and scFv8H9D4 in 3-fold molar and equimolar excess respectively. In conclusion, we have demonstrated that scFv-8H9D4 fully retains the functional properties of the original MA-8H9D4. This smaller antibody fragment (Mr - 24 kDa) may serve as a starting point for the design of pharmacological compounds for the treatment of patients with increased PAI-1 levels.