Production, purification and selection of second generation recombinant antithrombins of therapeutic interest : applications to severe sepsis and extracorporal circulation – THERAT

Antithrombin, a double agent, anticoagulant and cytoprotective : The THERAT project the coagulation inhibitor, antithrombin (AT). In addition to inactivate thrombin (factor II activated or factor IIa of coagulation) that converts fibrinogen into fibrin, AT physiological role is much broader: this plasma protein, which belongs to the family of serpins (Serine Protease Inhibitor) inhibits also several serine proteases of coagulation, including activated FX (FXa). The EA4531, University Paris Sud (partner 2) is particularly involved in the study of structure-function relationship of AT to strengthen and extend the properties of the therapeutic product, which is the natural protein extracted from human plasma and marketed by LFB (partner 1).

Indeed, the reaction of inhibition of coagulation mediated by AT is significantly increased in the presence of heparin derivatives, which include heparan sulfate cellular that bind to AT by their pentasaccharide motive. This antigoagulante protein has also cytoprotective properties (anti-inflammatory activity and vasorelaxant) mediated through binding to heparan sulfates on the surface of cells. Thus there is a rational for the development of recombinant molecules derived from natural antithrombin but by decoupling the anticoagulant and cytoprotective activities of AT.
Indeed, anti-inflammatory properties of natural AT are observed in vitro only at high doses (250 IU / kg), that used in current practice would be likely to cause a hemorrhagic risk associated with the administration of supraphysiological doses of an anticoagulant protein.

THERAT project focuses therefore on recombinant AT-derived molecules ("variants") to be developed for the management of severe sepsis, but also to be used as an antidote for heparins, particularly in the context of extra-body circulation.

In this context, recombinant variants of AT devoid of anticoagulant activity and have an increased affinity for heparin, have recently been described {Bianchini, 2011} and which constitute the proof of concept of project THERAT. Its objective is to generalize the approach and develop two parallel strategies of random mutagenesis and directed variants of AT with high therapeutic potential, and then test them in vitro and in vivo (EA 3859, University of Angers, partner 4), while implementing innovative methodologies of characterization (UMR CNRS 8612, partner 3) and purification (LFB BIOTECHNOLOGIES, partner 1).

Eventually, two clinical indications will be targeted by these new generation product : 1 - The neutralization of heparin derivatives used in the proceedings of extra-body circulation (CEC) 2 - The treatment of severe sepsis. For enhanced therapeutic benefit, selected AT variants will have to display a reduced anticoagulant activity and an increased binding to heparin compared to plasma AT, while having a satisfactory and PK being devoid of any immunogenic potential.