Specific reversal of factor Xa inhibitors with single domain antibodies

Introduction: Factor Xa inhibitors, such as rivaroxaban, apixaban, and edoxaban are frequently prescribed oral anticoagulants. If patients on factor Xa inhibitors experience hemorrhages, rapid infusion of reversal therapy can prevent hematoma expansion. Andexanet-alfa is the only approved reversal treatment for factor Xa inhibitors. A major drawback of andexanet-alfa is its cross-reactivity with tissue factor pathway inhibitor (TFPI) and heparan sulphate-antithrombin complexes, eliminating the physiological anti-coagulant protection of treated patients and thereby increasing their thrombosis risk. We have developed single domain antibodies (sdAbs) for highly specific reversal of edoxaban, apixaban and rivaroxaban.

Method: Specific sdAbs were selected with phage display technology from a phage library that was created from B cell mRNA obtained from immunized lamas exposed to edoxaban, apixaban and rivaroxaban. We quantified the effectiveness of anti-edoxaban, anti-apixaban and anti-rivaroxaban sdAbs by assessing thrombin generation (TG, both Calibrated Automated Thrombogram and ST-Genesia), factor Xa activity and coagulation induced platelet activation, in plasma spiked with edoxaban, apixaban and rivaroxaban. Furthermore, anti-edoxaban sdAb was evaluated in plasma of humans on edoxaban treatment and in mice treated with edoxaban.

Results: Our study demonstrates that anti-edoxaban, anti-apixaban and anti-rivaroxaban sdAbs effectively reverse TG inhibition by edoxaban, apixaban and rivaroxaban, respectively. Specifically, TG lag time was 6 minutes when 300 nM edoxaban was added to plasma, compared to 2 minutes in untreated plasma and in plasma with both 300 nM edoxaban and 300 nM anti-edoxaban sdAb ([Fig 1A and 1B]). Similarly, the peak thrombin levels in plasma decreased from 274 nM to 54 nM upon spiking with 300 nM edoxaban, but were fully restored to 273 nM with the addition of 300 nM anti-edoxaban sdAb ([Fig 1]A and fig [1]C). Furthermore, anti edoxaban could reverse the effects of edoxaban on the staR Xa test ([Fig 1D]) and it could restore edoxaban induced prolonged tail bleeding time in mice ([Fig 1E]). None of the sdAbs gave a hyper-thrombotic response.​​ Furthermore, factor Xa activity in plasma samples spiked with edoxaban was completely reversed by anti-edoxaban sdAb and anti-edoxaban sdAb also restored TG in plasma from patients treated with edoxaban. In addition to the anti-edoxaban sdAbs, anti-apixaban sdABs completely reversed the effects of apixaban on TG and anti-rivaroxaban sdAbs reversed the effects of rivaroxaban on TG (data not shown).

Fig 1  Complete reversal of edoxaban by anti-edoxaban single domain Antibodies in thrombin generation (fig 1A-C), staR DOAC factor Xa test (fig 1D) and tail bleeding time (fig 1E).

Conclusion: This study highlights the potential of sdAbs as a new class of factor Xa inhibitor reversal treatment, emphasizing their role in managing bleeding complications associated with factor Xa inhibitors.