ADAMTS13 fusion constructs with dual activity against VWF: a novel strategy for iTTP.

Background

Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening thrombotic microangiopathy. iTTP pathophysiology is based on severe ADAMTS13 deficiency caused by autoantibodies, leading to accumulation of ultra-large von Willebrand factor (VWF) multimers and microvascular thrombosis. Engineered ADAMTS13 variants reduce autoantibody binding. But some display impaired VWF-cleaving activity, whereas others such as glycan engineered variants maintain proteolytic function.

Objectives

To investigate a single-domain antibody (aVWF sdAb) targeting the A1 domain of VWF in an active conformation and two dual-action fusion constructs (MDTCS-aVWF and MDTCSm-aVWF) linking this sdAb with either a truncated ADAMTS13 (MDTCS) or an autoantibody-resistant mutant (MDTCSm).

Methods

Binding of aVWF to active VWF and the VWF A1 domain was assessed using ELISA-based assays. Proteolytic activity was tested using FRETS-VWF73, VWF multimer and beads-on-a-string assays. VWF-platelet interactions were measured by light transmission aggregometry and flow cytometry. Autoantibody resistance was evaluated using FRETS-VWF73 assays in the presence of plasma from iTTP patients.

Results

The aVWF sdAb selectively bound active VWF and inhibited platelet-VWF interaction in solution, without impairing platelet adhesion to collagen-bound VWF. Both MDTCS-aVWF and MDTCSm-aVWF retained high proteolytic activity against VWF and reduced platelet-VWF interactions. Notably, MDTCSm-aVWF demonstrated enhanced proteolytic activity and superior resistance to iTTP autoantibodies.

Conclusion

Fusion construct MDTCSm-aVWF, combining an enzymatically active, autoantibody-resistant ADAMTS13 variant with a VWF conformation-specific inhibitory sdAb, offers a novel alternative therapeutic approach for iTTP.