Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1

Katherine A Vousden,Clare Murray,David C Lowe,Martin Strain,Lynne A Murray,Philip R Mallinder,James A Huntington,Matthew A Sleeman,Anja Pomowski,Brian Naiman,Trevor Wilkinson,Bojana Popović ,I De Mendez ,Alan Carruthers ,Dwayne Johnson ,Philip M Newton ,Jane R Connor ,T Lundqvist ,Patrick Dufner,Tristan J Vaughan

doi:10.1038/s41598-019-38842-x

Abstract

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor.

Highlights

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin
The major physiological role of PAI-1 is to block the conversion of plasminogen to plasmin by tissue-type plasminogen activator and urokinase-type plasminogen activator[4]
PAI-1 is synthesised in the active conformation, which is characterised by the accessibility of its reactive centre loop (RCL) to protease binding[12,14]

Summary

Introduction

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. MEDI-579 inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). The number after ‘P’ indicates the position of the residue N-terminal to the scissile bond; the prime indicates residues C-terminal to the scissile bond Interaction of this bait region with the active site of either tPA or uPA in a 1:1 stoichiometric complex results in cleavage of the P1-P1′ bond and extensive structural re-arrangement, characterised by the insertion of the N-terminal portion of the RCL into β-sheet A and the www.nature.com/scientificreports/. After recognition of the RCL by a protease (magenta, centre), the protease is irreversibly translocated to the opposite pole of PAI-1 and trapped as a covalent complex (right)

Methods

Results

Conclusion