title: Elucidation of the paratope of scFv-8H9D4, a PAI-1 neutralizing antibody derivative.
abstract: Interfering with increased levels of plasminogen activator inhibitor-1 (PAI-1) might offer new therapeutic strategies for a variety of cardiovascular diseases. Inactivation of PAI-1 can be accomplished by a number of monoclonal antibodies (MA), including MA-8H9D4. In a previous study, a single-chain variable fragment (scFv-8H9D4) was cloned and found to have the same properties as the parental MA-8H9D4. In the present study, we identified the residues of scFv-8H9D4 that contribute significantly to the paratope. The complementarity determining region 3 from the heavy (H3) and the light (L3) chain were analysed through site-directed mutagenesis. Out of twelve mutations, only four residues appeared to contribute to the paratope. The affinity of scFv-8H9D4-H3-L97D for PAI-1 was 38-fold decreased (K(A) = 4.8 +/- 0.2 x 10(7) M(-1) vs. 1.8 +/- 0.7 x 10(9) M(-1) for scFv-8H9D4) whereas scFv-8H9D4-H3-R98Y did not bind to PAI-1. The affinities of scFv-8H9D4-L3-Y91S and scFv-8H9D4-L3-F94D for PAI-1 were 9- and 5-fold reduced, respectively, whereas the combined mutation resulted in an 86-fold decreased affinity (K(A) = 2.1 +/- 0.2 x 10(7) M(-1)). In accordance with the affinity data, these mutants had no, or a reduced, PAI-1 inhibitory capacity, confirming that these four particular residues form the major interaction site of scFv-8H9D4 with PAI-1. In combination with the three-dimensional structure, these data contribute to the rational design of PAI-1 neutralizing compounds.
authors.affiliation: Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Verbeke
authors.forename: Ann
authors.initials: A
authors.lastname: Gils
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Stassen
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
mesh_terms: D000915:Antibody Affinity / Q000235:genetics; D001666:Binding Sites, Antibody / Q000276:immunology*; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology*
chemical_list: D022801:Complementarity Determining Regions; D007135:Immunoglobulin Variable Region; D017395:Plasminogen Activator Inhibitor 1