Elucidation of the paratope of scFv-8H9D4, a PAI-1 neutralizing antibody derivative

Koen Verbeke; Ann Gils; Jean-Marie Stassen; Paul J. Declerck

doi:10.1055/s-0037-1613545

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2003; 89(01): 74-82
DOI: 10.1055/s-0037-1613545

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Elucidation of the paratope of scFv-8H9D4, a PAI-1 neutralizing antibody derivative

Koen Verbeke

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium

,

Ann Gils

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium

,

Jean-Marie Stassen

²Boehringer Ingelheim Pharma KG, Biberach, Germany

^*Current address: Thromb-X, Leuven, Belgium

,

Paul J. Declerck

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium

› Author Affiliations

Abstract

Summary

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 × 10⁷ M^–1 vs. 1.8 ± 0.7 × 10⁹ 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 × 10⁷ 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.

Keywords

PAI-1 - PAI-1 inhibitor - antibody - scFv - paratope

Full Text

References

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