Factor VIII Mimetic Rescue of Hemophilia B Causing Factor IX Variants

Background The intrinsic Xase enzyme complex is responsible for the rate-limiting step of sustained coagulation. It is composed of the protease, activated coagulation factor IX (FIXa), and the cofactor, FVIIIa, bound on phospholipid membranes; deficiencies in the activity of the intrinsic Xase result in hemophilia B (HB) or hemophilia A (HB), respectively. The FVIII-mimetic emicizumab has been widely adopted for prophylaxis for HA patients, in part because of the convenience of subcutaneous administration. Prophylaxis for HB patients, however, still requires frequent intravenous FIX administration. Within the Xase, complex, FVIIIa binding allosterically activates FIXa and enhances FX substrate binding, though the molecular details of the FVIIIa/FIXa interaction remain incompletely understood. We hypothesize that the hemostatic activity of select HB-causing mutations due to dysfunctional FVIIIa/FIXa interactions may be rescuable by FVIII-mimetics such as emicizumab.

Aims To determine the spectrum of HB-causing missense FIX variants that have rescuable procoagulant activity with FVIII-mimetics and clarify the interaction between FIXa and FVIIIa at an amino acid level.

Methods Recombinant FIX variants were expressed from transiently transfected HEK293 cells. Procoagulant activity of recombinant FIX variant and patient samples were assayed by either aPTT assay, one-stage FIX activity, thrombin generation assay (TGA), and rotation thromboelastography (ROTEM).

Results The ability of the FVIII-mimetic emicizumab to rescue the procoagulant activity of HB-causing FIX variants was screened using a modified one-stage FIX activity assay. To account for the high clotting activity of emicizumab in aPTT-based assays, the FIX activity of the HB-causing variants was compared against the activity of wild-type (WT) FIX in the presence of an equal concentration of emicizumab. FVIII-mimetic rescuable HB-causing variants were selected if their activity relative-to-FIX-WT increased at least twofold with the addition of emicizumab. About 20% of FIX variants evaluated to-date appear to be rescuable with emicizumab, including K5E, L6S, D47E, D332Y, R333Q, R338P, P368T, E387G, E387K, and I397T.

To further define the procoagulant rescue of these FIX variants, the amount of thrombin generated by these FIX variants in HB plasma was measured with and without emicizumab and compared to FIX-WT with the same emicizumab concentrations. Representative thrombograms are shown in the Figure. The addition of emicizumab increased the peak thrombin generation of the rescuable HB-causing variants 2-6 fold relative-to-FIX-WT. In contrast, emicizumab had no effect on the amount of thrombin generated by the catalytically inert FIX-S365A (S195A in chymotrypsin numbering).

We also obtained samples from two moderate HB patients with the I397T missense variant; these patients have between 1-3% normal FIX activity and a moderate bleeding phenotype. We observed that emicizumab could normalized the prolonged aPTT of their plasma and improved the ROTEM clot time of their whole blood in a dose-dependent manner.

Conclusions The procoagulant activity of select HB-causing missense FIX variants can be rescued with therapeutics amounts of emicizumab. These variants likely have a dysfunctional interaction with FVIIIa cofactor, which is circumvented in the presence of a FVIII-mimetic. Importantly, such rescuable variants are identified throughout the FIXa protein, highlighting the range of amino acids involved in the FIXa/FVIIIa interaction. Identified rescuable variants likely has dysfunctional FVIIIa-binding (K5E, L6S) and/or disturbed allosteric activation (E387K, I397T). FVIII mimetics may be an alternative prophylactic hemostatic therapy for HB patients with such rescuable variants.

Pishko:Biomarin: Other: Advisory Board; Sanofi Genzyme: Research Funding. George:Pfizer: Consultancy; AskBio Therapeutics: Other: Receives licensing fees ; Bayer: Consultancy; Spark: Consultancy; Biomarin: Consultancy; Intellia: Consultancy; Avrobio: Other: Data safety monitoring committee; STRM.Bio: Other: Scientific advisory board. Samelson-Jones:Cabaletta: Patents & Royalties; Biomarin: Consultancy; Amarna: Membership on an entity's Board of Directors or advisory committees; GeneVentiv: Membership on an entity's Board of Directors or advisory committees; Frontera: Consultancy.