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.
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
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K
Verbeke
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A
Gils
P J
PJ
Declerck
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