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

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.

Molecular mechanism of two nanobodies that inhibit PAI‐1 activity reveals a modulation at distinct stages of the PAI‐1/plasminogen activator interaction

“…Thus, Nb42 possesses a novel mechanism of PAI‐1 neutralization, that is, destabilizing both the Michaelis complex and acyl‐enzyme bound to the initial binding site on PAI‐1, via interference with exosite interactions that do not block the inhibitory reaction between PAI‐1 and PAs or change the SI. This mechanism is considerably different from previously described antibodies MEDI‐579, MA‐56A7C10, and MA‐42A2F6, which also bind directly to residues in the RCL, thereby blocking the accessibility of the P1‐P1′ bond for the PA active site.…”

“…47 interactions that do not block the inhibitory reaction between PAI-1 and PAs or change the SI. This mechanism is considerably different from previously described antibodies MEDI-579, 28 MA-56A7C10, 54 and MA-42A2F6, 54 which also bind directly to residues in the RCL, thereby blocking the accessibility of the P1-P1′…”

“…Compared with these ligand‐bound structures, the epitopes of PAI‐1 neutralizing mAbs and Nbs have been mapped to different regions of the PAI‐1 molecule . Although several molecular mechanisms of PAI‐1 neutralization by mAbs were discovered, only one crystal structure of PAI‐1 complexed with an inhibitory antibody fragment was reported . The structure containing the Fab fragment of the neutralizing antibody MEDI‐579 reveals that the fragment binds the RCL from P2 to P3′, including the reactive center P1‐P1′ residues, and the adjacent exosite for binding the so‐called 37‐loop of PAs.…”

“…Beyond the very few structures of PAI‐1 complexes with small molecule or peptide inhibitors and only one recent structure of PAI‐1 in complex with an antibody Fab fragment, no structures of PAI‐1/Nb complexes have been described. Here, we report the first atomic structures of two stabilized active PAI‐1 mutants, PAI‐1‐W175F and PAI‐1‐N150H‐K154T‐Q301P‐Q319L‐M354I (PAI‐1‐stab), each in a triple complex with Nb42 and Nb64 as well as PAI‐1‐stab in complex with Nb42.…”

“…23 According to biochemical characterization, Nb64 switched the PAI-1/PA interaction to the substrate pathway, whereas Nb42 appeared to interfere with the initial steps of the PAI-1/PA complex formation. 23 Beyond the very few structures of PAI-1 complexes with small molecule or peptide inhibitors [25][26][27] and only one recent structure of PAI-1 in complex with an antibody Fab fragment, 28 no structures of PAI-1/Nb complexes have been described. Here, we report the first atomic structures of two stabilized active PAI-1 mutants, PAI-1-W175F and PAI-1-N150H-K154T-Q301P-Q319L-M354I (PAI-1-stab), each in a triple complex with Nb42 and Nb64 as well as PAI-1-stab in complex with Nb42.…”