Inhibition of plasminogen activator inhibitor-1: antibody fragments and their unique sequences as a tool for the development of profibrinolytic drugs.
Physiological inhibition of plasminogen activator inhibitor-1 (PAI-1) might improve the prevention and treatment of various cardiovascular diseases. To date, a variety of monoclonal antibodies that neutralize PAI-1 have been generated. The current study presents the cloning, expression and characterization of four single-chain variable fragments (i.e. scFv-33B8, scFv-33H1F7, scFv-35A5 and scFv-55F4C12) from the corresponding PAI-1 neutralizing monoclonal antibodies. Surprisingly, affinity constants of scFv-33B8, scFv-33H1F7 and scFv-55F4C12 for PAI-1 (KA = 1.4 +/- 0.2 x 1010 m-1, 3.7 +/- 0.1 x 109 m-1, 1.0 +/- 0.2 x 109 m-1, respectively) were only 2- to 4-fold lower compared to those of the respective monoclonal antibodies (MAs). In contrast, scFv-35A5 exhibited a 6250-fold decrease in affinity (KA = 3.2 +/- 0.8 x 106 m-1 vs. 2.0 +/- 0.8 x 1010 m-1 observed for MA-35A5) with a concomitant absence of functional effects on PAI-1 activity. Evaluation of the dose-response curves of the PAI-1 neutralizing effect of the other scFvs revealed a shift towards slightly higher concentrations (in line with the small decrease in affinity) eventually resulting in a similar maximum effect as the corresponding MAs (i.e. 92 +/- 2%, 34 +/- 3% and 66 +/- 5% PAI-1 inhibition for scFv-33B8, scFv-33H1F7 and scFv-55F4C12, respectively). In conclusion, the sequence information of the scFvs allows to humanize MAs with PAI-1 inhibiting properties whereas the scFv constructs serve as an excellent starting point for structure based drug design, both aiming at the reduction of cardiovascular diseases.
Verbeke
K
K
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
Gils
A
A
Declerck
P J
PJ
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D005343:Fibrinolytic Agents; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology*
D005343:Fibrinolytic Agents; D007128:Immunoglobulin Fragments; D017395:Plasminogen Activator Inhibitor 1; C055966:immunoglobulin Fv