Prolonged Half-Life and Improved Recovery of Recombinant Factor IX-XTEN Fusion Proteins in Hemophilia B Mouse Model
Authors: van der Flier Arjan , Liu Zhiqian Lucy , Liu Zhan , Mercury Oblaise , Ismail Ayman , Seth-Chhabra Ekta , Kulman John , Schellenberger Volker , Light David R. , Peters Robert 
Published: October 2019

INTRODUCTION

Prophylactic treatment for hemophilia B patients is the therapy of choice to improve quality of life and minimize annual bleeding rates and damage to joints. A new generation of extended half-life (EHL) FIX replacement products has been generated to improve patient care by reducing treatment burden. The factors include the currently marketed rFIXFc, as well as rFIX attached to PEG or albumin. The latter two are in clinical trials. All FIX preparations are administered by intravenous dosing, which can be particularly challenging for young patients and patients with limited venous access; these difficulties are reduced, but not eliminated, by the less frequent dosing achieved with EHL rFIX therapies. In the current study, we evaluate an XTEN-recombinant protein technology in order to develop EHL rFIX-XTEN molecules that are suitable for both acute treatment, as well as prophylactic subcutaneous dosing in hemophilia B, and could potentially further reduce the burden of treatment. XTEN are unstructured polypeptide sequences that consist of a limited set of natural amino acids (Pro, Ala, Gly, Glu, Ser, Thr). Fusion of XTEN to proteins alters its hydrodynamic properties and reduces the rate of clearance and degradation of the fusion protein. These XTEN fusion proteins are produced using recombinant technology, without the need for chemical modifications, and degraded by natural pathways.

MATERIALS AND METHODS

Recombinant FIX and FIXFc molecules were expressed as the natural Arg338Leu (R388L, Padua) variant with improved activity. XTEN polypeptides are fused to either the C-terminus of rFIX or inserted into the EGF2 domain or activation peptide (AP) domain of rFIX or rFIXFc. The fusion proteins were prepared by transient expression in human HEK293 cells followed by affinity purification. Hemophilia B (HemB) mice were dosed by either intravenous or subcutaneous injection with a single bolus of 50 or 200 IU/kg of the rFIX- or rFIXFc-fusion proteins. Plasma activity levels were determined over time using a FIX activated partial thromboplastin time assay (aPTT). PK parameters were determined using non-compartmental modeling with Phoenix WinNonlin 6.2.1 (Pharsight).

RESULTS

Insertion of XTEN sequences with increasing length (42, 72, 144 or 288 amino acids long) at either C-terminus of rFIX-R338L or in the AP domain showed a size dependent increase in plasma recovery up to 60% following intravenous bolus dosing. Combinations of XTEN insertions in the EGF2 or AP domain with Fc-mediated half-life extension in rFIXFc-R338L, extended the half-life as well as increased the plasma recovery. The AUC/D for rFIX-CT-XTEN.288 and rFIXFc-AP-XTEN.72 were 8.5 and 14.5 fold improved when compared to intravenously dosed rFIX, respectively.

Following a subcutaneous dose of either rFIXFc-AP-XTEN.72 or rFIX-CT-XTEN.288, we observed 28 and 40-fold improved AUC/D; 15- and 30-fold improved Cmax/D and 3-fold increased bioavailability. When compared to rFIXFc the improvement in pharmacokinetic parameters was 6- and 9-fold improved AUC/D; 3- and 10-fold Cmax/D and 1.5- and 2-fold improved bioavailability for FIXFc-AP-XTEN.72 and rFIX-CT-XTEN.288, respectively.

Taken together, subcutaneous dosing of rFIX-CT-XTEN.288 and rFIXFc-AP-XTEN.72 in HemB mice showed improved AUC/D when compared to intravenous dosing of rFIXFc.

CONCLUSIONS

rFIXFc-AP-XTEN.72 and rFIX-CT-XTEN.288 show greatly improved subcutaneous pharmacokinetics in HemB mice compared to both rFIX and rFIXFc. These promising preclinical subcutaneous dosing data in HemB mice suggests the potential of once weekly or every two weeks prophylactic subcutaneous dosing of FIX-XTEN molecules in patients. In addition, the molecules have potential for acute treatment by intravenous dosing. Further studies are ongoing to address the efficacy and allometric scaling in preclinical animal models.

Disclosures

van der Flier: Biogen: Employment. Liu:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership, Honoraria, Research Funding. Mercury:Biogen: Employment. Ismail:Biogen: Employment. Seth-Chhabra:Biogen: Employment, Equity Ownership, Honoraria, Patents & Royalties, Research Funding. Kulman:Biogen: Employment. Schellenberger:Amunix Operating Inc: Employment. Light:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.