OC 29.4 - Development of recombinant antibodies targeting the coagulation factor XIII-B subunit for research and therapeutic use

Background: Plasma coagulation factor XIII (FXIII) is a heterotetramer composed to two A and two B subunits (FXIII-A2B2). The FXIII-A subunits, once activated, catalyze covalent crosslinking in the fibrin network, whereas the FXIII-B subunits stabilize the FXIII-A subunits in circulation. The FXIII-B subunits also mediate the binding of FXIII-A2B2 to fibrinogen. Interestingly, this binding interaction enhances FXIII activation rate, which in turn modulates the retention of red blood cells in venous thrombi, and consequently, thrombus size. Therefore, therapeutic disruption of the FXIII-fibrinogen interaction represents a potential approach to reduce venous thrombus burden.

Aims: Develop and characterize recombinant anti-FXIII-B antibodies.

Methods: Recombinant FXIII-B was expressed in mammalian cells and used as a capture antigen in phage display with a library of phage expressing fragment antigen binding domains (Fabs, Figure 1). Binding affinities were measured using bio-layer interferometry.

Results: Recombinant FXIII-B bound fibrinogen, as expected (KD=46.9 nM). Fab-Phage selection yielded seven clones that bound immobilized FXIII-B with nanomolar affinity (KD=2.7-22.2 nM). Stronger affinities were observed with solution-phase FXIII-B (KD=0.7-11.3 nM), likely due to the avidity of dimeric FXIII-B in solution. None of the Fabs were competitive for fibrinogen binding to FXIII-B, but three of these clones bound plasma-derived FXIII-A2B2, although with reduced affinity relative to FXIII-B alone. Conversion of these three Fabs to bivalent IgGs dramatically improved the binding affinity for recombinant FXIII-B (KD < 100 pM). These three clones in IgG format were still non-competitive for fibrinogen binding. Consistent with this observation, epitope binning revealed that these clones bound the same epitope on FXIII-B.

Conclusion(s): Fab-Phage display is an effective strategy to generate new recombinant anti-FXIII-B antibodies. These antibodies represent tools to support studies investigating FXIII-B structure and function. Efforts using different selection approaches and phage display libraries to identify antibodies that block FXIII-fibrinogen binding and modulate FXIII function in coagulation are ongoing.