Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate.
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.
Debrock
S
S
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit, Leuven, Belgium.
Sironi
L
L
Declerck
P J
PJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D051379:Mice; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology* / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D017395:Plasminogen Activator Inhibitor 1