Anti-TFPI Single-Domain Antibodies: Novel Rebalancing Therapies for Hemophilia and Other Rare Bleeding Disorders

Tissue factor pathway inhibitor (TFPI) is an important determinant of thrombin generation in patients with deficiency in factor (F)VIII, FIX, and FXI. As such, anti-TFPI monoclonal antibodies such as concizumab have been developed to treat hemophilia A (HA) and B (HB).The objective of this study is to generate single-domain antibodies (sdAbs) as a novel class of pharmacological agents blocking TFPI and increasing thrombin generation in the plasma of patients with severe HA, HB, and FXI deficiency.A large synthetic library of sdAbs was generated and selected by phage-display on recombinant human TFPIα (rhTFPIα). Anti-TFPI sdAbs were screened on their ability to promote thrombin generation. Their binding to the Kunitz (K1, K2, K3) domains of TFPIα were measured by enzyme-linked immunosorbent assay. Their effects on the inhibitory activity of rhTFPIα toward FXa and TF/FVIIa complex were tested in purified systems. Thrombin generation was measured on plasmas from patients with severe HA, HB, or FXI-deficiency spiked with the anti-TFPI sdAbs.A total of 14 out of 188 screened sdAbs specifically bound to rhTFPIα, and two of them (26E5 and 26E8) targeted the K1 domain of TFPI and increased thrombin generation in the plasma of patients with HA. Both sdAbs impaired the ability of rhTFPIα to inhibit FXa and TF/FVIIa, in the absence and presence of their physiological modulators (protein S and FXa, respectively). These sdAbs efficiently increased thrombin generation in HB and FXI-deficient plasmas.Our large synthetic library could be used for readily generating diverse and functionally relevant anti-TFPI sdAbs that may be therapeutically attractive.