New targeted therapy for hemophilia

In this issue of Blood, Jiang et al¹ describe the development of a new monoclonal antibody that binds with very high affinity to activated protein C (APC) and selectively inhibits its anticoagulant activity to restore hemostasis in preclinical models of hemophilia.

Maintenance of hemostasis is central to the prevention of thrombosis and bleeding. Diminished thrombin generation and clot formation arising from clotting factor deficiencies, such as hemophilia, disrupt this delicate balance and promote bleeding. Factor replacement is the standard of care for people with hemophilia in wealthy countries, but alternative hemostatic products are required if inhibitors to replacement factors develop. Although the factor VIII (FVIII) mimetic bispecific antibody emicizumab has enhanced the treatment of patients with hemophilia A with inhibitors, the need for alternative therapies for individuals with hemophilia B and other rare bleeding disorders has stimulated the generation of a plethora of new prohemostatic agents that target different facets of the coagulation system. Of these, novel therapies that can safely attenuate endogenous anticoagulant pathways may represent a solution to “rebalance” hemostasis in individuals with inherited or acquired bleeding disorders. To this end, antibody-mediated inhibition of tissue factor pathway inhibitor or aptamer-mediated suppression of antithrombin has already demonstrated positive outcomes in clinical trials with patients with hemophilia.

Inhibition of the protein C pathway also represents an attractive target based on its central role in the dynamic regulation of thrombin generation.² Plasma protein C is activated by thrombin bound to its anticoagulant receptor, thrombomodulin, which is abundantly expressed on the vessel wall. This complex, in turn, converts protein C into its activated form, APC, in a process accelerated by protein C binding to the endothelial protein C receptor. APC with its cofactor protein S then degrades activated procoagulant cofactors FVa and FVIIIa to restrict further thrombin generation (see figure). APC also exists in minute quantities in plasma (∼40 pM), and attenuation of its anticoagulant properties may help promote thrombin generation in individuals with bleeding disorders. Several creative approaches have already been developed to achieve APC inhibition. SerpinPC, a recombinant α1-antitrypsin variant with enhanced specificity for APC, promotes hemostasis by inhibiting APC anticoagulant activity and is currently being evaluated in phase II clinical trials in patients with hemophilia B.³ In addition, SuperVa, an APC-resistant FVa variant, effectively promotes thrombin generation in preclinical bleeding models.⁴ Furthermore, inhibition of the APC anticoagulant cofactor protein S promotes hemostasis in hemophilia plasma and preclinical hemophilia models.⁵ Most recently, monoclonal antibodies (mAbs) targeting APC have shown significant promise in directly blocking FVa proteolysis by APC to promote hemostasis.^6,7 

A critical safety concern for therapies that inhibit anticoagulant pathways is that they do not “overbalance” hemostasis to promote thrombosis. Therapeutic approaches that inhibit APC may have an additional safety consideration, namely, the inadvertent inhibition of important APC properties that are distinct from its role in regulating hemostasis. APC possesses complex anti-inflammatory signaling functions that are principally mediated by proteolysis of protease-activated receptor 1 (PAR1) on both endothelial and peripheral immune cells (see figure). Importantly, genetic deficiency or antibody inhibition of APC can exacerbate both acute and chronic inflammatory disease in preclinical models. Although not evaluated to date, it is reasonable to assume that such a possibility could arise in patients with hemophilia receiving APC-blocking therapies that inhibit all proteolytic activity. The latest iterations of APC-targeting monoclonal antibodies have therefore sought to selectively attenuate APC anticoagulant properties without influencing the capacity to promote APC signaling.^6,7 Helpfully, exosites on the APC surface necessary for isolating specific APC functions have already been extensively characterized.

In this issue, Jiang et al describe the development and characterization of an engineered humanized mAb (SR604) designed to target an APC exosite located within the APC protease domain that is critical for anticoagulant activity but dispensable for PAR1-dependent anti-inflammatory signaling. SR604 was based on a previous murine mAb (HAPC1573) that bound APC but required high doses to restore hemostasis in animal hemophilia models. Evaluation of mAb-APC binding affinity by surface plasmon resonance showed that SR604 possessed a 60-fold higher affinity for APC than its parent antibody. This translated into enhanced hemostatic efficacy in ex vivo plasma experiments, in which SR604 dose-dependently shortened clotting times in various coagulation factor-deficient plasmas with APC generation. In vivo, SR604 limited tail bleeding from hemophilic mice to a similar extent as recombinant FVIII and reduced joint bleeding and arthropathy in a mouse knee injury model performed in hemophilic mice. Importantly, the authors confirmed that SR604 did not exert a similar inhibition of APC signaling activity on endothelial cells, showing that SR604 did not affect PAR1-mediated APC protection of endothelial cell barrier function from thrombin-induced permeability. Intriguingly, SR604 accelerated APC proteolysis of cytotoxic extracellular histones, another crucial anti-inflammatory APC function during acute infection, suggesting SR604 may also represent a useful tool for better understanding the mechanistic basis of this distinct anti-inflammatory activity. To further demonstrate the lack of SR604-mediated interference in endogenous APC anti-inflammatory properties, the authors administered SR604 to hemophilic mice that had been exposed to a sublethal lipopolysaccharide (LPS) dose and showed that, in contrast to an anti-protein C monoclonal antibody that blocks APC generation, SR604 binding to endogenous APC did not provoke increased mortality in response to LPS challenge. These data support the reduced impact of SR604 on APC anti-inflammatory activity compared with its potent inhibition of anticoagulant activity. However, further studies will be required for more granular assessment of whether SR604 modulates other noncanonical APC signaling functions^8,9 and to determine whether SR604 impacts APC signaling in chronic inflammatory disease settings in which endogenous APC is also protective.¹⁰ 

Although pending evaluation in clinical trials, the prohemostatic properties, limited off-targeted toxicities, and favorable pharmacokinetics described in this study suggest SR604 represents an exciting addition to the growing range of prohemostatic nonfactor therapies and underscore the potential of precision therapies that target selected protein C pathway functions. This study also provides a salient example of the continued refinement of approaches to rebalance hemostasis safely, which is anticipated to expand further the therapeutic options available to individuals with hemophilia and other rare bleeding disorders.

Conflict-of-interest disclosure: The author declares no competing financial interests.