Abstract: 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.

Abstract: D assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D′D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D′D3 forms the binding site for factor VIII. The crystal structure of monomeric D′D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D′ with its TIL′ and E′ modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL′ domain of D′ distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.

Abstract: Abstract 2266 Recombinant Factor IX Fc fusion (rFIXFc) was designed to be a long-lasting version of recombinant FIX that has the potential to provide less frequent dosing. This may be applicable to prophylaxis and on-demand therapy of hemophilia B and for control of bleeding during surgery. The potency of the rFIXFc drug product, as well as the commercially available recombinant factor IX, BeneFIX®, is assigned by a one-stage clotting assay. The thrombin generation assay (TGA), a global hemostasis assay that monitors the amount of active thrombin produced in patient plasma after recalcification, represents a useful indication in the evaluation of coagulation capacity of hemophilic plasma. When equal units of rFIXFc and BeneFIX, as determined by the one stage assay, were spiked into hemophilic plasma and their coagulation capacity was assessed by the TGA, BeneFIX generated 2-fold higher peak thrombin and significantly left-shifted thrombin curve relative to rFIXFc in the presence of limiting tissue factor and 4 μM phospholipids. In an assay control without tissue factor triggering, BeneFIX demonstrated considerable thrombogenic activity, whereas rFIXFc was essentially inactive. Since BeneFIX was observed to have a markedly higher level of FIXa impurity than rFIXFc in a factor IXa ELISA, and since factor IXa protease is known to be highly thrombogenic in an in vivo model, we hypothesized that the enhanced in vitro thrombin generation profile of BeneFIX may be due to the presence of excess factor IXa. BeneFIX was incubated overnight with a serine protease active site blocker, EGR-chloromethyl ketone, and dialyzed by extensive buffer exchange. The FIXa-blocked BeneFIX showed very similar thrombin generation profile (ETP, peak thrombin, time course and slope) to rFIXFc, confirming the role of FIXa in thrombin generation by BeneFIX. To quantify the amount of active factor IXa in BeneFIX and rFIXFc, a plasma-derived factor IXa (pFIXa) standard curve was constructed by spiking increasing concentrations of factor IXa (0–100 pM) into human factor IX-deficient plasma in the presence of 4 μM phospholipids. Prior to starting the measurement, 5 nM thrombin was added to the assay in order to improve sensitivity. A dose response was observed with a detection limit as low as 0.5 pM pFIXa in FIX-deficient plasma. BeneFIX, FIXa-blocked BeneFIX and rFIXFc of equal potency (1 IU/mL by the one-stage clotting assay) generated thrombin responses comparable to 20 pM, 1 pM and 2 pM pFIXa, respectively, indicating the amount of FIXa present in each FIX product. In a regular thrombin generation assay triggered with limiting tissue factor, 1 IU/mL rFIXFc supplemented with 20 pM pFIXa demonstrated an equal peak thrombin and velocity index to 1 IU/mL BeneFIX. These data suggest that: 1) Minor amounts of FIXa protease in a FIX drug product (0.1%) can trigger significant thrombin generation in global hemostasis assays 2) Thrombin generation assay could be used to evaluate FIXa level in FIX products with high sensitivity (0.5 pM FIXa per IU/ml FIX) 3) the higher peak thrombin and shortened time course in the thrombin generation profile for BeneFIX relative to rFIXFc is caused entirely by the presence of factor IXa in the drug product 4) Discounting the rFIXa impurities in these drug products, BeneFIX and rFIXFc have equivalent in vitro thrombin generation activity per unit of FIX activity. Disclosures: Buyue: Biogen Idec Hemophilia: Employment. Seth Chhabra:Biogen Idec Hemophilia: Employment. Wang:Biogen Idec Hemophilia: Employment. Sommer:Biogen Idec Hemophilia: Employment.

Abstract: 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.

Abstract: In addition to its ability to accelerate filament assembly, which is common to formins, INF2 is a formin protein with the unique biochemical ability to accelerate actin filament depolymerization. The depolymerization activity of INF2 requires its actin monomer-binding WASP homology 2 (WH2) motif. In this study, we show that INF2 is peripherally bound to the cytoplasmic face of the endoplasmic reticulum (ER) in Swiss 3T3 cells. Both endogenous INF2 and GFP-fusion constructs display ER localization. INF2 is post-translationally modified by a C-terminal farnesyl group, and this modification is required for ER interaction. However, farnesylation is not sufficient for ER association, and membrane extraction experiments suggest that ionic interactions are also important. The WH2 motif also serves as a diaphanous autoregulatory domain (DAD), which binds to the N-terminal diaphanous inhibitory domain (DID), with an apparent dissociation constant of 1.1 μM. Surprisingly, the DID-DAD interaction does not inhibit the actin nucleation activity of INF2; however, it does inhibit the depolymerization activity. Point mutations to the DAD/WH2 inhibit both the DID-DAD interaction and depolymerization activity. Expression of GFP-INF2 containing these DAD/WH2 mutations causes the ER to collapse around the nucleus, with accumulation of actin filaments around the collapsed ER. This study is the first to show the association of an actin-assembly factor with the ER.

Abstract: Introduction: Scientific evidence increasingly suggests that, for many patients with severe hemophilia A, targeting higher sustained factor VIII (FVIII) levels is required to improve protection from bleeds, preserve joint health, and advance closer to the goal of a functional cure and health equity (Skinner MW, et al. Haemophilia. 2020). However, achieving high sustained factor activity levels with once-weekly dosing is not possible with currently approved FVIII therapies due to the FVIII half-life ceiling imposed by endogenous von Willebrand factor (VWF). Efanesoctocog alfa (rFVIIIFc-VWF-XTEN; BIVV001) is a novel fusion protein designed to decouple recombinant FVIII (rFVIII) from endogenous VWF and thereby overcome the VWF-imposed half-life ceiling (Chhabra ES, et al Blood. 2020). Once-weekly efanesoctocog alfa (50 IU/kg) has a geometric mean half-life of 41 hours and provides high sustained FVIII activity levels in the normal to near-normal range ( & gt;40%) for 3-4 days post dose and 10% at Day 7 (Lissitchkov T, et al. Blood. 2019). Therefore, efanesoctocog alfa has the potential to offer extended bleed protection with less frequent dosing in patients with severe hemophilia A (Konkle B, et al. N Engl J Med. 2020). In the Phase 1/2a and Phase 1 studies (EXTEN-A single-dose and repeat-dose, respectively), efanesoctocog alfa was well tolerated in previously treated adults with severe hemophilia A and no safety concerns were identified (Konkle B, et al. N Engl J Med. 2020; Lissitchkov T, et al. Blood. 2019). The aim of this post hoc analysis is to evaluate the relationship between endogenous VWF antigen levels and efanesoctocog alfa half-life and clearance in patients with severe hemophilia A from the single- and repeat-dosing studies. Methods: A total of 40 previously treated adult males (N=16 and N=24, respectively) with severe hemophilia A ( & lt;1 IU/dL [ & lt;1%] endogenous FVIII at screening) and ≥150 exposure days of prior FVIII treatment enrolled in the EXTEN-A single-dose (NCT03205163) and repeat-dosing (EudraCT No: 2018-001535-51)studies (Konkle B, et al. N Engl J Med. 2020; Lissitchkov T, et al. Blood. 2019). Subjects received either a single IV dose of 25 IU/kg (n=7) or 65 IU/kg (n=9) efanesoctocog alfa in EXTEN-A (Konkle B, et al. N Engl J Med. 2020) or 4 once-weekly doses of either 50 IU/kg (n=10) or 65 IU/kg (n=14) efanesoctocog alfa in the repeat-dosing study (Lissitchkov T, et al. Blood. 2019). Each study assessed safety and tolerability as its primary objective, with pharmacokinetics (PK) evaluated as a secondary objective (Konkle B, et al. N Engl J Med. 2020; Lissitchkov T, et al. Blood. 2019). All subjects with evaluable PK profiles were included in this post hoc analysis. The half-life and clearance of efanesoctocog alfa were evaluated as a function of pre-dose endogenous VWF antigen levels. VWF antigen levels were also assessed at various time points after dosing through end of study. Linear correlations were calculated using Pearson's correlation coefficient. Results : Of 40 enrolled subjects, mean (range) age was 39 (19-63) years. Mean (range) pre-dose VWF antigen levels were 151% (74%-297%; n=14) and 128% (49%-265%; n=24) for the single- and repeat-dose studies, respectively. Individual patient antigen levels of endogenous VWF were relatively stable over time, from screening and pre dose through 336 hours post dose and end of study (Figure 1A). Similar results were observed for VWF:RCo levels. A total of 37 subjects were eligible for inclusion in the pooled correlation analyses; 3 subjects had missing values and were excluded from analysis. Efanesoctocog alfa half-life showed no correlation with VWF antigen levels (R 2=0.0007; P=0.88) (Figure 1B). A similar result was observed for clearance of efanesoctocog alfa, which showed no correlation with VWF antigen levels (R 2=0.0493; P=0.19). Conclusions: Endogenous VWF levels are unaffected during and after treatment with single- or repeat-dosing of efanesoctocog alfa in previously treated patients with severe hemophilia A. Furthermore, the half-life and clearance of efanesoctocog alfa are independent of endogenous VWF levels. This is consistent with previous findings from preclinical data and supports the continued evaluation of efanesoctocog alfa as a VWF-independent rFVIII in ongoing Phase 3 clinical trials in hemophilia A (XTEND-1, NCT04161495; XTEND-Kids, NCT04759131; XTEND-ed, NCT04644575). This analysis was funded by Sanofi and Sobi. Figure 1 Figure 1. Disclosures Staber: Bayer, CSL Behring, Sanofi, Takeda: Membership on an entity's Board of Directors or advisory committees. Lissitchkov: Catalist: Other: Principal Investigator of Clinical Trials; Grifols: Other: Principal Investigator of Clinical Trials; Bayer: Other: Principal Investigator of Clinical Trials. Konkle: BioMarin Pharmaceutical Inc.: Other: Data and safety monitoring; Sigilon Therapeutics: Honoraria; CSL Behring: Other: Data and safety monitoring; Genentech USA Inc.: Honoraria. Shapiro: OPKO: Research Funding; Prometric BioTherapeutics: Research Funding; Sangamo: Other: Advisory board fees, Research Funding; Takeda: Research Funding; Sigilon Therapeutics: Other: Advisory board fees, Research Funding; Glover Blood Therapeutics: Research Funding; Genentech: Other: Advisory board fees, Research Funding, Speakers Bureau; Octapharma: Research Funding; Pfizer: Research Funding; Novo Nordisk: Other: Advisory board fees, Research Funding, Speakers Bureau; Novartis: Research Funding; Kedrion Biopharma: Research Funding; BioMarin: Research Funding; Bioverativ (a Sanofi company): Other: Advisory board fees, Research Funding; Daiichi Sankyo: Research Funding; Agios: Research Funding. Quon: Orthopaedic Institute for Children: Current Employment. Kulkarni: Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire/Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hamilton: Sobi: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Chhabra: Sanofi: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Katragadda: Sanofi: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Altincatal: Sanofi: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Willemze: Sanofi: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Dumont: Sanofi: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Ragni: Takeda Therapeutics: Membership on an entity's Board of Directors or advisory committees; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioverativ (Sanofi): Membership on an entity's Board of Directors or advisory committees; BioMarin Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Alnylam (Sanofi): Membership on an entity's Board of Directors or advisory committees; University of Pittsburgh: Research Funding.