Novel Therapeutics for Bleeding Disorders Research Overview



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Emicizumab is now used to prevent bleeding in hemophilia A, but prophylaxis for hemophilia B (HB) patients still requires frequent intravenous factor IX administration. We will attempt to develop emicizumab or similar bi-specific antibodies for HB therapy by identifying HB-causing FIX variants amenable to emicizumab rescue, evaluating this approach in preclinical studies, and developing a clinical trial evaluating this strategy for HB prophylaxis.

At a molecular level, FVIIIa serves as a cofactor for FIXa to activate FX, which is the rate-limiting step of sustained coagulation. We hypothesize that emicizumab can treat HB caused by FIX variants with dysfunctional FIXa/FVIIIa interactions. We have conducted initial screening of three HB-causing FIX variants with amino acid substitutions within the FVIIIa-binding site (R338P, E387K, and I397T), and one outside this motif (R248Q), as a negative control. We observe that FVIIIa-binding site mutations are rescued with emicizumab, resulting in mild HB FIX activity.

In contrast, the non-FVIIIa-binding site variant (R248Q) does not respond to emicizumab. Emicizumab similarly increases the FIX activity in HB patient plasma with the I397T variant to the mild HB range, while it does not change the FIX activity in plasma from a cross-reactive material negative (CRM-) HB patient. These results suggest that FVIII-mimicking bispecific antibodies may improve the bleeding in HB patients with dysfunctional FIXa/FVIIIa interactions. Plasma from HB patients will be screened for emicizumab-rescuing activity. As >50% of severe and moderate HB-causing variants are due to missense mutations, this approach may be applicable to hundreds of HB patients.

We propose to develop monoclonal antibodies (mAbs) that bind and protect factor V from activated protein C (APC)-mediated inactivation and promote coagulation in the context of hemophilia or other rare bleeding disorders (RBDs). APC is a major anticoagulant and protecting FV from inactivation is expected to enhance coagulation. There is a strong rationale for this approach as APC-resistant FV yields a procoagulant phenotype (e.g., FV-Leiden; R506Q mutation). Individuals and mice with FV-Leiden and hemophilia exhibit reduced bleeding and require fewer FVIII infusions. When FV-Leiden is present in hemophilia A (HA) plasma, there is enhanced thrombin generation.

We generated a panel of mAbs against FV and screened for ones that protect it from APC inactivation. Using a thrombin generation assay, the addition of APC to HA plasma reduced thrombin generation to baseline levels while addition of mAb GB5 restored thrombin generation. These data suggest that the mAb binds FV and protects it from APC inactivation and demonstrates this may work for hemophilia.

We initiated preliminary in vivo experiments to test the effectiveness of this mAb to promote coagulation. Since the mAbs do not bind mouse FV, we infused mice FV with human FV. Clot formation was evaluated using an intravital laser injury animal model infused with hFV in the presence or absence of GB5. Fibrin formation assessed was more robust in the presence of GB5 following injury compared to the no-antibody control. These preliminary data suggest GB5 is protecting FV/FVa from APC, leading to enhanced clot formation in vivo.

Mechanisms contributing to factor VIIIa inactivation are: 1) rapid A2-domain dissociation and 2) APC cleavage; A2 dissociation is widely thought to be the predominant mechanism of FVIIIa regulation. We hypothesize that combining mutations that inhibit APC cleavage and diminish A2-domain dissociation will produce additive or synergistic enhanced FVIII hemostatic function for hemophilia A (HA) therapeutic benefit.

Mutations were combined with FVIII-QQ to generate FVIII-QQ-VV, a protein resistant to APC cleavage and A2 dissociation; this approach inhibits both mechanisms of FVIIIa regulation and has not been described. We aim to generate novel FVIII variants resistant to A2-domain dissociation drawing from studies of FVIII’s homologous protein FV. We identified the FVIII homologous residues and will generate the FVIII variant proteins and determine if they are resistant to inactivation. The proteins will be purified, characterized, and lead candidates evaluated in the context in AAV gene addition. We will also perform exploratory studies using gene-editing techniques to knock-in FVIII cDNA downstream of the endogenous FVIII or albumin promoter.

Prior pre-clinical attempts of gene editing for HA have thus far been stymied by low expression; this problem may be solved by a gain of function FVIII variant. Further, a gene-editing approach may overcome limitations of current gene-addition efforts in HA and HB. Particulary, current gene addition strategies that confer hepatocyte expression from an episomal donor transgene will not impart lifelong efficacy when treating infants and children without re-dosing, which is currently unattainable. Thus, gene editing, if successful, may ultimately replace current gene addition approaches and extend gene therapy efforts to children.


Wilhelm AR, Parsons NA, Samelson-Jones BJ, Davidson RJ, Esmon CT, Camire RM, George LA. Activated protein C has a regulatory role in factor VIII function. Blood. 2021 May 6;137(18):2532-2543. PMID: 33512448

Chau J, Sternberg A, Pishko A, George L, Samelson-Jones B. Improved Procoagulant Activity of Hemophilia B Causing Dysfunctional Factor IX Variants with Factor VIII Mimetics [abstract]. Accessed Nov. 7, 2022.