Hemophilia results from an inherited imbalance of hemostasis caused by a deficiency in a coagulation factor: factor VIII (FVIII) in hemophilia A and factor IX (FIX) in hemophilia B. These disorders, which affect approximately one to two boys per 10,000 and one per 25,000 at birth, respectively, predispose patients to a bleeding diathesis. Treatment usually relies on replacement of the deficient factor, using plasma-derived or recombinant concentrates. This approach nonetheless has limitations: the need for frequent intravenous access and, above all, the development of inhibitory antibodies that neutralize the therapeutic factor in up to 30% of patients with hemophilia A and about 3% of those with hemophilia B. Patients who have developed such inhibitors represent an ongoing therapeutic challenge that recent therapies—non-factor replacement and in vivo gene therapy—seek to address.
The authors propose an original strategy based on single-domain antibody fragments (single-domain antibodies, or sdAbs) derived from the llama. Rather than replacing the missing factor, these fragments aim to rebalance hemostasis by inhibiting antithrombin, one of the anticoagulant proteins that naturally restrain the coagulation cascade. Following immunization of a llama, monovalent sdAbs were assembled into multivalent formats: a bi-paratopic fragment named KB-AT-23, linking two monovalent antibodies through peptide bonds. These constructs were first evaluated in vitro and then administered to mice, either directly as a protein or via a liver-tropic adeno-associated viral vector of serotype 8 (AAV8), with expression placed under the control of a hepatocyte-specific promoter.
In vitro, the multivalent fragments efficiently blocked antithrombin activity and restored thrombin generation potential in FVIII-deficient plasma. Administered as a protein to hemophilia A mice, the bi-paratopic fragment significantly reduced blood loss in a model of acute hemorrhagic injury (tail-clip procedure). Packaged in the AAV8 vector, this same fragment, durably expressed in the liver, proved safe and weakly immunogenic. Most importantly, this sustained hepatic expression corrected the bleeding phenotype in mice with both hemophilia A and hemophilia B, including in the presence of inhibitory antibodies directed against the therapeutic coagulation factor.
This work demonstrates that single-domain antibodies directed against antithrombin can rebalance hemostasis independently of FVIII or FIX, whether delivered as a protein or through hepatic gene therapy. The efficacy observed in murine models carrying inhibitors represents the most notable contribution of this approach, which dispenses with the deficient factor itself.