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Factor VIII (FVIII) replacement products enable comprehensive care in hemophilia A. Treatment goals in severe hemophilia A are expanding beyond low annualized bleed rates to include long-term outcomes associated with high sustained FVIII levels. Endogenous von Willebrand factor (VWF) stabilizes and protects FVIII from degradation and clearance, but it also subjects FVIII to a half-life ceiling of ∼15 to 19 hours. Increasing recombinant FVIII (rFVIII) half-life further is ultimately dependent upon uncoupling rFVIII from endogenous VWF. We have developed a new class of FVIII replacement, rFVIIIFc-VWF-XTEN (BIVV001), that is physically decoupled from endogenous VWF and has enhanced pharmacokinetic properties compared with all previous FVIII products. BIVV001 was bioengineered as a unique fusion protein consisting of a VWF-DʹD3 domain fused to rFVIII via immunoglobulin-G1 Fc domains and 2 XTEN polypeptides (Amunix Pharmaceuticals, Inc, Mountain View, CA). Plasma FVIII half-life after BIVV001 administration in mice and monkeys was 25 to 31 hours and 33 to 34 hours, respectively, representing a three- to fourfold increase in FVIII half-life. Our results showed that multifaceted protein engineering, far beyond a few amino acid substitutions, could significantly improve rFVIII pharmacokinetic properties while maintaining hemostatic function. BIVV001 is the first rFVIII with the potential to significantly change the treatment paradigm for severe hemophilia A by providing optimal protection against all bleed types, with less frequent doses. The protein engineering methods described herein can also be applied to other complex proteins.

Introduction: Prophylactic treatment for hemophilia has demonstrated clear benefit over on demand treatment in hemophilic patients. Alprolix (rFIXFc) is the first of a new generation of long acting clotting factors we made to reduce the frequency of intravenous infusions (Powell, NEJM, 2013). Nevertheless, intravenous injections can be particularly challenging for young patients and patients with limited venous access and are time consuming. We are developing a novel rFIXFc fusion protein, using XTEN recombinant technology, which is suitable for prophylactic subcutaneous dosing in hemophilia B. In the current studies we explore the in vivo bleeding efficacy of subcutaneously dosed rFIXFc-XTEN in hemophilia B mouse model. Methods: recombinant rFIXFc-XTEN was produced in HEK293 cells and affinity purified. The subcutaneous PK profile of rFIXFc-XTEN was compared to that of intravenously dosed rFIXFc in hemophilia B mice. The prolonged and acute bleeding efficacies were evaluated in HemB mouse tail-vein transection (TVT) and tail-clip bleeding models. Results and conclusions: Subcutaneous dosing of rFIXFc-XTEN shows a tmax around 20 hours post dosing in mice, and improved plasma activity levels compared to similar (IU/kg) intravenously dosed rFIX or rFIXFc. Using the TVT bleeding model in HemB mice we show that at 72 hours post dosing, subcutaneously dosed rFIXFc-XTEN has improved in vivo efficacy compared to intravenously dosed rFIXFc at all tested doses. Similarly, acute efficacy testing in the HemB mouse tail-clip bleeding model showed improved efficacy of intravenously dosed rFIXFc-XTEN compared to rFIXFc. These data support the potential of once weekly or less frequent subcutaneous prophylactic dosing of rFIXFc-XTEN in humans. Disclosures Liu: biogen: Employment, Equity Ownership. van der Flier:Biogen: Employment, Equity Ownership. Ismail:Biogen: Employment, Equity Ownership. Simpson:Biogen: Employment, Equity Ownership. Kurt:Biogen: Employment, Equity Ownership. Schellenberger:Amunix: Employment, Equity Ownership. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder.

Background: A variety of approaches have been used to extend the half-life of human factor IX (FIX) in the circulation, including PEGylation and recombinant fusion of either the Fc domain of IgG or albumin to the C-terminus of FIX. Introduction of XTENs, unstructured hydrophilic polypeptides of defined amino acid composition and low immunogenic potential, represents a promising alternative approach for generating FIX variants with improved pharmacokinetic properties. Moreover, the hydrophilic properties of XTEN have the potential to increase the solubility of FIX and thereby render it suitable for subcutaneous administration, which is currently an unmet need. Because XTEN is introduced into FIX by DNA recombination, the location, length, composition and number of XTEN modifications can be readily varied, and impact of these modifications on the activity and clearance of FIX can be evaluated. Aim: To identify sites in FIX that can accommodate the introduction of XTENs without abrogating FIX activity and apply this approach to both otherwise non-modified FIX and a recombinant FIX-Fc fusion protein. Methods: The highly active FIX Padua variant (R338L) was used as a scaffold to counter FIX activity loss due to reduced activity caused by the introduction of XTENs. XTEN insertion within the Gla domain was avoided due to the essential role of the Gla domain in anchoring FIX to phospholipid surfaces and subendothelial type IV collagen. XTEN insertion sites were selected by analysis of available FIX structures in the Protein Data Bank in conjunction with the following criteria: 1) calculated accessible surface area, 2) solvent accessibility assessed by hydrogen/deuterium exchange mass spectrometry (H/DX-MS), 3) exclusion of sites within defined secondary structural elements, 4) preference for positions with significant inter-species protein sequence variability, and 5) exclusion of sites proximal to known hemophilia B mutations. A 42-residue XTEN element (AE-42) was inserted at sites selected by using these criteria or fused at the C-terminus of FIX. FIX activities of these variants were evaluated in conditioned medium of transfected HEK293 cells. Longer XTENs (AE-72, -144 and -288) were then similarly tested at sites shown to be permissive for AE-42 insertion. Finally, based on results obtained with these single XTEN variants, FIX variants with multiple XTEN insertions of varying lengths and at representative sites were evaluated for FIX activity. Results: Based on the criteria described above, a total of 33 sites in FIX were selected and evaluated by insertion of AE-42. Of these, two in EGF domain 2 (EGF2), one in the EGF2-activation peptide (AP) linker region, four in the AP, and four in the catalytic domain, including the C terminus, were identified as permissive sites by FIX activity assay. Only sites in AP and sites at or close to the C-terminus of FIX tolerated longer XTENs (AE-144, AE-288 or AE-864). FIX activity detected in conditioned medium inversely correlated with the length of XTEN introduced. Four representative sites in FIX were selected to generate a combinatorial library of 79 FIX variants. Three groups, FIX with a single XTEN, FIX with dual XTENs and FIX-Fc with a single XTEN insertion, showed detectable activity, while combination of insertion/fusion at three or more sites abolished FIX activity. Conclusions: Several permissive sites for XTEN insertion are present in FIX and select combinations of XTEN insertions variants retain FIX activity. Active FIX-XTEN variants identified here are candidates for pharmacokinetic characterization in hemophilia B mice. Disclosures Liu: Biogen: Employment. van der Flier:Biogen: Employment. Bardan:Biogen: Employment. Ding:Amunix: Employment. Schellenberger:Amunix Operating Inc: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

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.

All currently marketed Factor VIII (FVIII) molecules are administered intravenously (IV) for the treatment of hemophilia A (HemA). Conventional FVIII prophylaxis requires a dosing interval of three times per week to every other day. This frequent dosing regimen necessitates repeated venous access and is associated with complications such as secondary infection in children with a venous port/catheter. More recently, extended half-life variants of FVIII have been shown in clinical trials to decrease the dosing interval to twice weekly or less frequent IV dosing, which reduces, but does not eliminate, the burden of treatment. A FVIII molecule with further prolonged half-life and subcutaneous (SQ) delivery potential could significantly relieve the treatment burden for HemA patients and improve the adherence rate to FVIII prophylaxis. Recombinant FVIIIFc-VWF-XTEN has been shown to not bind endogenous VWF, and is able to achieve a 4-fold extension of half-life in hemophilia A mice compared to conventional FVIII, well beyond the approximately 2-fold half-life extension limit demonstrated by other long-acting FVIII variants that bind endogenous VWF. It comprises of two polypeptide chains: 1) a single chain B-domain deleted FVIIIFc-XTEN chain with a XTEN polypeptide inserted at the B-domain region of native FVIII sequence, and 2) a VWF D'D3-XTEN-Fc chain xtend one that n TEN fragemnt o FVIII prophylaxis.ntial rity of the patients depending on the half-life of the FVIII molecule. with a second XTEN polypeptide inserted between D'D3 domain and Fc. The rFVIIIFc-VWF-XTEN protein was produced in HEK293 cells and affinity purified using VIIISelect resin. The pharmacokinetic (PK) profiles of intravenously (IV) and subcutaneously (SQ) administered rFVIIIFc-VWF-XTEN were compared to those of rFVIII in HemA mice. The duration of the in vivo efficacy of rFVIIIFc-VWF-XTEN post-SQ delivery was assessed in a HemA mouse tail vein transection (TVT) bleeding model. After intravenous dosing in HemA mice, we observed a linear PK profile for rFVIIIFc-VWF-XTEN within the therapeutic dose range (25, 50, 100 IU/kg). The half-life of IV-administered rFVIIIFc-VWF-XTEN was about 37 h, which is more than 4-fold longer than that of rFVIII. In addition, animals that received 25 IU/kg of rFVIIIFc-VWF-XTEN treatment retained 5% of normal FVIII activity at 120 h post-dosing, which suggests the potential for full protection from spontaneous bleeding in this animal model. When delivered subcutaneously, the bioavailability of rFVIIIFc-VWF-XTEN was 20%, a significant increase compared to the bioavailability of rFVIII (less than 1%). Starting at 24 h post-dosing, subcutaneous administration of rFVIIIFc-VWF-XTEN achieved plasma FVIII levels that were equal to or greater than those attained with rFVIII delivered intravenously at the same dose. In addition, greater than 5% of normal circulating FVIII level was observed at 96 h post SQ administration of rFVIIIFc-VWF-XTEN with a 100 IU/kg dose, which provided 80% protection on survival in mice subjected to tail vein transection injury. These results suggest that rFVIIIFc-VWF-XTEN could enable less frequent FVIII replacement treatment compared to rFVIII even when administered subcutaneously. The VWF independence of rFVIIIFc-VWF-XTEN enables a 4-fold increase in circulating half-life compared to that of rFVIII. Also, the addition of D'D3 domains and the two XTEN insertions dramatically increases subcutaneous bioavailability to 20%, compared to less than 1% with conventional FVIII. These unique properties of rFVIIIFc-VWF-XTEN make it a potential candidate for both IV and SQ treatments for hemophilia A. Disclosures Drager: Biogen: Employment, Equity Ownership. Patarroyo-White:Biogen: Employment, Equity Ownership. Chao:Biogen: Employment, Equity Ownership. Ismail:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership. Holthaus:Biogen: Employment. Chhabra:Biogen: Employment, Equity Ownership. Kulman:Biogen: Employment. Schellenberger:Amunix Operating Inc: Employment. Liu:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

The development of inhibitors to replacement plasma factors in hemophilia is an ongoing clinical complication. Bypass therapies, such as recombinant factor VIIa (rFVIIa), have emerged as important alternative on-demand strategies for hemophilic patients with inhibitors to treat spontaneous bleeds and prevent bleeding during surgery. However, general prophylaxis strategies for hemophilia inhibitor patients are lacking. An attractive approach for effective prophylaxis and on-demand treatment includes engineering potent rFVIIa variants with prolonged pharmacokinetics. Because the primary mechanism of action of rFVIIa is thought to occur on the platelet surface, we combined rFVIIa with platelet-targeting and XTEN half-life extension technologies to improve its pharmacokinetic profile and procoagulant activity. Platelet-targeting was achieved by recombinant fusion of an antibody fragment which binds the human alpha IIb integrin. Half-life extension was achieved by fusion of an XTEN polypeptide which increases the hydrodynamic radius, and therefore half-life, of rFVIIa. We have shown that these rFVIIa modifications improve the pharmacokinetics and efficacy of rFVIIa in vivo in humanized alpha IIb transgenic hemophilia A mice. The goal of the current study was to evaluate and compare thrombin generation and fibrin formation kinetics in hemophilic platelet-rich plasma in the presence of platelet-targeted rFVIIa-XTEN or rFVIIa. To achieve this, platelet-rich plasma from normal human donors was treated with an inhibitory factor VIII antibody to model hemophilia A and spiked with doses of platelet-targeted rFVIIa-XTEN or rFVIIa. Reactions were triggered with low tissue factor and recalcification. Thrombin generation (n=9) was monitored by calibrated automated thrombography and fibrin formation (n=7) was monitored optically on a plate reader. Non-linear regression analysis of dose responses was used to determine EC50 values for each parameter for each donor. Platelet-targeted rFVIIa-XTEN increases the rate and peak of thrombin generation with 2- to 6-fold lower EC50 values (peak and rate, respectively) than rFVIIa. These data were confirmed by thrombin generation in platelet-rich plasma from 1 hemophilia A donor and 1 hemophilia B donor which demonstrated similar responses to platelet-targeted rFVIIa-XTEN with 2.5- to 5-fold lower EC50 values (peak and rate, respectively) than rFVIIa. Notably, platelet count-dependent changes in thrombin generation activity were similar between platelet-targeted rFVIIa-XTEN and rFVIIa. Fibrin formation analysis indicated platelet-targeted rFVIIa-XTEN increases the rate and time to plateau of fibrin formation with 5- to 13-fold lower EC50 values (rate and time to plateau, respectively) than rFVIIa. Analysis of fibrin network structure by confocal microscopy indicated platelet-targeted rFVIIa-XTEN increases fibrin network density in platelet-rich plasma clots. Together, these data show platelet-targeted rFVIIa-XTEN has more procoagulant activity than rFVIIa by supporting more thrombin generation and faster fibrin formation and suggest our approach has the potential to be an effective alternative for the treatment and prevention of bleeds in hemophilia patients with inhibitors. Disclosures Aleman: Biogen: Employment, Equity Ownership. Kistanova:Biogen: Employment, Equity Ownership. Moore:Biogen: Employment, Equity Ownership. Schellenberger:Amunix Operating Inc: Employment. Peters:Biogen: Employment. Salas:Biogen: Employment, Equity Ownership.

INTRODUCTION More than 95% of circulating clotting factor VIII (FVIII) exists in a non-covalent complex with von Willebrand Factor (VWF). While VWF stabilizes and protects FVIII from its clearance pathways, it also subjects FVIII to VWF-mediated clearance. Thus, interaction with VWF imposes a limitation on the extent of FVIII half-life extension achieved by current technologies (Fc fusion, PEGylation etc.). Recombinant FVIIIFc-VWF-XTEN (rFVIIIFc-VWF-XTEN) is a novel fusion protein, consisting of the FVIII binding D'D3 domains of VWF fused to a single chain rFVIIIFc (scFVIIIFc). Appending the domains of VWF to FVIII provides the protection and stability of endogenous VWF, while avoiding the limitation imposed by VWF clearance. Besides D'D3 domains, it also contains two XTEN linkers. XTEN is an unstructured polypeptide consisting of six amino acids repeats (Gly, Ala, Pro, Glu, Ser, Thr). Fusion of XTEN to a protein reduces the rate of clearance and degradation of the fusion protein. In rFVIIIFc-VWF-XTEN, one XTEN linker replaces the B-domain of FVIII and other is attached to the D'D3 domains. In preclinical studies, this protein has shown >4-fold prolonged half-life and similar in vivo acute efficacy compared to rFVIII. In the current study, we examined the impact of various modifications on the in vitro plasma stability of rFVIIIFc-VWF-XTEN protein. MATERIALS AND METHODS rFVIIIFc-VWF-XTEN is a fusion protein which is expressed as a dual chain molecule. One chain expresses the D'D3 domains linked to a Fc monomer through an XTEN linker. This polypeptide is co-expressed with a single chain rFVIIIFc monomer to generate a dimer, via the disulfide-bond between the Fc domains. To assess the in vitro plasma stability, fusion proteins were expressed in HEK293 cells, purified and incubated with plasma from FVIII KO (Hem A) or FVIII/VWF DKO mice, for various time periods at 37 degree centigrade. After the desired incubation time, plasma stability of the recombinant proteins was determined by FVIII chromogenic activity assay. Results and Conclusions rFVIIIFc-VWF-XTEN fusion protein showed significantly enhanced in vitro plasma stability compared to rFVIII. In FVIII KO plasma, rFVIII started losing activity by 4 hours, and by 24 hours it lost more than 80% of its activity. The decline in activity was more pronounced and rapid when rFVIII was incubated with FVIII/VWF DKO plasma, mainly due to the absence of protection provided by VWF. Conversely, in the case of rFVIIIFc-VWF-XTEN, there was no significant drop in activity even after 6 hours (in both FVIII KO and DKO plasma). By 24 hours, only 10-15% activity reduction was observed in FVIII KO plasma and about a 35% decrease in DKO plasma. Further studies were conducted to evaluate various parameters which contributed to the improved stability of this fusion protein. Our results suggest that there are multiple factors which contribute to the overall stability of rFVIII-VWF-XTEN protein. These include: presence of covalently attached D'D3 domains, enhanced stability of single chain FVIII isoform used in the fusion protein and presence of the XTEN linker in the B-domain of FVIII. These data suggest that superior plasma stability of this novel fusion protein might be a contributing factor to its prolonged in vivo half-life and efficacy. Disclosures Seth Chhabra: Biogen: Employment, Equity Ownership. Moore:Biogen: Employment, Equity Ownership. Furcht:Biogen: Employment, Equity Ownership. Holthaus:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Schellenberger:Amunix Operating Inc: Employment. Kulman:Biogen: Employment. Salas:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.