A molecular jewel for hemophilia A treatment

In this issue of Blood, Seth Chhabra et al describe the engineering of a new therapeutic chimera composed of B domain–deleted (BDD) factor VIII (FVIII), FVIII-binding domain of von Willebrand factor (VWF), Fcγ1 fragment, and XTEN polypeptides.¹  The molecule, referred to as rFVIIIFc-VWF-XTEN or BIVV001, has a prolonged half-life that is independent from endogenous VWF and is hemostatically competent.

Hemophilia A is a rare X-linked inherited hemorrhagic disorder resulting from deficiencies in procoagulant FVIII. Prevention or treatment of bleeding episodes is achieved by use of FVIII-derived products or bypassing agents. Expectedly, no FVIII-surrogate drug is as efficacious as FVIII in performing all of the hemostatic functions of FVIII. Hence, primary prophylaxis with FVIII products remains the treatment of choice for patients with severe hemophilia A.²  However, the use of therapeutic FVIII meets with 2 major hurdles: (i) the short half-life of the molecule (ie, 12 to 19.7 hours) that imposes repeated administration of FVIII to ensure optimal joint health and patients’ quality of life, and (ii) the immunogenicity of the molecule that leads to the development of neutralizing anti-FVIII immunoglobulin G (IgG) in ∼30% of patients. With an estimated half-life of 33 to 34 hours in nonhuman primates, BIVV001 represents a breakthrough in the field.

Previous attempts to generate long-lasting FVIII molecules have exploited the Fc fusion or PEGylation technologies. Although the clinical use of extended half-life FVIII products has led to an at least 30% reduction in the frequency of IV injections,²  half-life extension is limited (1.3- to 1.7-fold on average) and, in the case of Fc-fused products, is far from the expected 3-week-long half-life of human IgG. One of the reasons is that Fc-fused and PEGylated FVIII products retain their capacity to bind VWF, the chaperon of FVIII.³  VWF protects FVIII from proteolytic degradation and unwanted activation, controls its catabolism by preventing its binding to catabolic receptors, transports FVIII to the bleeding sites, and reduces FVIII immunogenicity by reducing its endocytosis by antigen-presenting cells. The other side of the coin however is that VWF dictates FVIII half-life, notably by acting as a sink and driving the elimination of VWF-bound FVIII upon interaction with VWF-specific catabolic receptors. As a result, the half-life of FVIII-derived molecules able to bind VWF is not expected to exceed that of VWF (ie, 9 to 15 hours). This limitation is levered in BIVV001.

BIVV001 is the outstanding achievement of rational protein engineering (see figure). In the molecule, modification of the D′D3 region of VWF by mutation of cysteine residues prevents the formation of D′D3 dimers, whereas the cis-addition of the VWF propeptide D1D2 ensures optimal folding of D′D3 and highest affinity for FVIII. Fusion of the BDD-FVIII moiety and of the modified D1D2D′D3 moiety to the Fc fragment of human IgG1 further stabilizes the association between D′D3 and FVIII by virtue of the disulfide bonds between the Fc fragments. It also favors the binding of the molecule to the neonatal Fc receptor (FcRn), which guides the intracellular routing of the endocytosed molecules away from lysosomal degradation pathways and promotes recycling to the circulation.⁴  Importantly, insertion of a 288-amino-acid-long polypeptide (referred to as XTEN) between the A2 and A3 domains of FVIII and of a 144 XTEN between the D′D3 and Fc domains confers increased solubility and reduces in vivo clearance. Insertion of the FVIII acidic region 2 (a2) thrombin cleavage site between the D′D3 and Fc domains ensures optimal release kinetics of activated FVIII. Last, removal of the PPVLKRHQR sequence N-terminal to the A3 domain eliminates the furin cleavage site between the heavy and light chains of FVIII, thus fostering production of FVIII as a single polypeptide. Together, iterative steps of rational molecular improvements have generated a technological jewel with normal procoagulant FVIII activity that does not bind to endogenous VWF and is endowed with a truly extended half-life in both mice and nonhuman primates. Of note, the achieved half-life is shorter than that of a fusion D′D3-Fc,⁵  suggesting that a deeper understanding of FVIII biology should allow further improvement of its pharmacokinetics in the future. Of course, the half-life of BIVV001 is also much shorter than that of emicizumab, a recombinant bispecific antibody that mimics the cofactor function of activated FVIII. In contrast to emicizumab, however,⁶  BIVV001 is expected to provide full protection under conditions of severe hemostasis challenge, such as trauma, major surgery, or strenuous physical activity.

Whether the increase in pharmacokinetics of BIVV001 is accompanied by a reduction in its immunogenicity remains to be determined. The predicted T-cell epitope PPVLKRHQR present in the C-terminal region of the B domain of the commercially available rFVIIIFc, Eloctate (patent CA2841066A1), has been removed in BIVV001. In addition, BIVV001 contains 2 XTEN polypeptides that, in essence, are devoid of T-cell epitopes and are claimed to reduce the immunogenicity of the molecules they are fused to.⁷  Furthermore, the binding of FVIII to the D′D3 domain of VWF reduces its endocytosis by antigen-presenting cells in vitro,⁸  and the presence of VWF in FVIII products was proposed to reduce FVIII immunogenicity in vivo.⁹  Taken together, this evidence suggests that the immunogenicity of BIVV001 should not be greater, and may be lower, than that of Eloctate (clinical trial #NCT02196207). Whether D′D3 alone may prevent FVIII uptake to a similar extent as the entire VWF molecule is unknown. Importantly, although the endocytosis of FVIII by antigen-presenting cells is necessary to initiate neutralizing anti-FVIII humoral immune responses, it is also a prerequisite to the induction of active FVIII-specific immune tolerance and to the maintenance of established immune tolerance. Future studies will need to investigate whether BIVV001 shows an altered immunological profile as compared with FVIII products with physiological affinities toward VWF. Will BIVV001 be able to induce FVIII-specific tolerance as we believe is spontaneously happening in ∼70% of severe patients during standard FVIII replacement therapy?¹⁰  Will BIVV001 allow maintenance of protective tolerance in hemophilia A patients who have developed active immune tolerance to therapeutic FVIII, either spontaneously or after immune tolerance induction by high-dose FVIII?