Single‐domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors

Abstract

Novel therapies for hemophilia, including non‐factor replacement and in vivo gene therapy, are showing promising results in the clinic, including for patients having a history of inhibitor development. Here, we propose a novel therapeutic approach for hemophilia based on llama‐derived single‐domain antibody fragments (sdAbs) able to restore hemostasis by inhibiting the antithrombin (AT) anticoagulant pathway. We demonstrated that sdAbs engineered in multivalent conformations were able to block efficiently AT activity in vitro, restoring the thrombin generation potential in FVIII‐deficient plasma. When delivered as a protein to hemophilia A mice, a selected bi‐paratopic sdAb significantly reduced the blood loss in a model of acute bleeding injury. We then packaged this sdAb in a hepatotropic AAV8 vector and tested its safety and efficacy profile in hemophilic mouse models. We show that the long‐term expression of the bi‐paratopic sdAb in the liver is safe and poorly immunogenic, and results in sustained correction of the bleeding phenotype in hemophilia A and B mice, even in the presence of inhibitory antibodies to the therapeutic clotting factor.

Skeletal muscle myosin and cardiac myosin attenuate heparin's antithrombin‐dependent anticoagulant activity

“…Inhibition of AT's anticoagulant activity can have a variety of effects. Multiple studies have shown the promising effect of inhibiting AT's anticoagulant pathway as a potential treatment for hemophilia, 40,41 with an RNA interference therapy currently being investigated in phase 3 clinical trials 42,43 . Because SkM and CM blunt UFH's and HS's enhancement of AT activity, it is possible that a better understanding of the multiple molecular interactions responsible for these effects might point toward the development of novel compounds that, like myosins, might also reduce AT potency by binding endogenous heparin/HS, thus affecting hemophilia‐related bleeding.…”

Laboratory monitoring of hemophilia A treatments: new challenges

“…51 The focus in this section will be on the thrombin generation assay. As illustrated in Figure 2, the addition of emicizumab, nanobodies against antithrombin, 52 or polyclonal antibodies against TFPI 53 has a strong stimulating effect on the thrombin generation profile in FVIII-deficient plasma. Thus, the presence of the procoagulant antibody emicizumab or the functional absence of antithrombin or TFPI results in increased thrombin peaks and the endogenous thrombin potential compared with FVIII-deficient plasma alone.…”

“…21 Primates were given an anti-FVIII antibody that specifically inhibits primate presence of anti-antithrombin nanobodies. 52 (C) Tissue factor-induced thrombin generation in the absence or presence of polyclonal anti-TFPI antibodies. 53 FVIII.…”

“…However, it is unclear if lower levels of FVIII would have been sufficient to stop the bleeding in this model. Based on recent data using nanobodies that block antithrombin activity in a tail clip model using FVIII-deficient mice, 52 it would be fair to assume that a reduction of antithrombin levels to 30% of normal corresponds to an FVIII-equivalence of at least 20% FVIII. have been used to estimate FVIII equivalence for nonfactor therapies.…”

“…In an alternative approach using anti-antithrombin nanobodies, inhibition of antithrombin activity appeared to correspond to an FVIII equivalence of $20%. 52 Regarding concizumab, a rabbit model for acquired hemophilia has been described. 65 Also in this model, an FVIII calibrator was unavailable.…”