OC 39.2 - Development of antibodies to disrupt FXIII/fibrinogen interactions for venous thrombosis prevention

Background: Plasma coagulation factor XIII (FXIII) is composed of two A and two B subunits forming a heterotetrameric zymogen (FXIII-A2B2). The FXIII-A subunits catalyze fibrin crosslinking to increase clot mechanical stability and prevent premature fibrinolysis, and the FXIII-B subunits mediate FXIII-A2B2 binding to fibrinogen to enhance the FXIII activation rate. Although complete FXIII deficiency causes bleeding, mouse models show reduced or delayed FXIII activation suppresses venous thrombosis (VT) by decreasing red blood cell retention to reduce clot mass without increasing bleeding.

Aims: Develop antibodies that disrupt FXIII binding to fibrinogen to decrease VT by delaying FXIII activation and reducing clot mass.

Methods: Antibodies against human FXIII-B were developed using a Fab-Phage display library. Candidates were selected for their ability to: i) disrupt FXIII-B binding to fibrinogen (biolayer interferometry and fibrinogen precipitation assays); ii) delay FXIII-A activation and fibrin crosslinking (immunoblotting and fibrin formation assays); and iii) reduce whole blood clot mass (clot contraction assays).

Results: Two antibodies (identified as Ab3 and Ab4) with subnanomolar affinities for FXIII-B disrupted binding of human FXIII-B to human fibrinogen in a purified system and prevented FXIII-B from co-precipitating with fibrinogen in normal pooled human plasma. Both Ab3 and Ab4 profoundly delayed FXIII-A activation and formation of high molecular weight crosslinked fibrin species and reduced the mass of contracted human whole blood clots by 25.5 and 27.8%, respectively. Ab3 also disrupted binding of mouse FXIII-B to mouse fibrinogen, delayed FXIII-A activation and formation of high molecular weight crosslinked fibrin species in mouse plasma, and reduced the mass of contracted mouse whole blood clots by 56.6%.

Conclusion(s): We have developed anti-FXIII-B antibodies that disrupt FXIII-B/fibrinogen interactions, delay FXIII-A activation and fibrin crosslinking, and reduce whole blood clot mass. These antibodies represent a novel antithrombotic strategy to decrease VT burden.