ALBERT

Description: Background: ACE910, asymmetric bispecific monoclonal antibodies to activated factor IX (IXa) and factor X, mimics the cofactor function of activated factor VIII (VIIIa) by modulating an optimal position on the tenase assembly. The estimated therapeutic range of ACE910 shows ~30% of thrombin generation in native tenase assembly, supporting that the structure on ACE910-mimicking tenase assembly is different from that on native tenase. Being close to physiological structure consisting from factor IXa, factor X, and factor VIIIa is important for potentiating the clotting function. We examined the effects of factor VIII subunits (light chain, heavy chain, A1 and A2, C2) on ACE910-tenase. Materials/Methods: The factor VIII light chain and heavy chain were isolated from EDTA-treated recombinant factor VIII following chromatography on SP- and Q- Sepharose columns. The A2 and A1 subunits were purified from thrombin-cleaved factor VIII heavy chain by Heparin-, SP- Sepharose columns. Purified factor Xa generation assays was examined with (i) factor VIII subunit (0-40 nM), ACE910 (10 µg/ml), phospholipid (PL) (40 µM), factor IXa (1 nM) and factor X (200 nM), (ii, iii) the A2 or heavy chain (40 nM), ACE910 (10 µg/ml), PL (40 µM), factor IXa and factor X (1 or 0-80 nM, and 0-300 or 200 nM, respectively). These mixtures were reacted for five minutes (i, ii) or one minute (iii). These assays were conducted at 37 °C. Results: (i) The factor Xa generation in ACE910-tenase complex in the absence of factor VIIIa was 10.1±2.2 nM. With the intact heavy chain and A2, amounts of factor Xa were increased dose-dependently, resulting in 1.3- and 1.2-fold increases, respectively. While, the light chain and A1 subunit failed to increase at all. (ii) Vmax for factor X in ACE910-tenase was 173.0±7.0 nM and Km was 31.2±3.9 nM. Vmax obtained with the heavy chain or A2 was 175.9±6.1 or 159.0±6.1 nM, whilst Km was 17.0±2.2 or 31.9±3.5 nM, respectively, indicating that the heavy chain enhanced the binding affinity for factor X in ACE910-tenase. (iii) Vmax for factor IXa in ACE910-tenase was 43.8±2.7 nM and Km was 36.9±4.8 nM. With the heavy chain or A2, Vmax was 46.8±3.0 or 45.0±3.1 nM, and Km was 36.4±3.0 or 32.1±4.9 nM, respectively, indicating that either the heavy chain or A2 did not enhance the catalytic activity and the binding affinity for factor IXa in ACE910-tenase. Conclusion: ACE910-tenase assembly seems to be close to physiological structure by the presence of intact heavy chain interacting with factor X. In addition, ACE910 may substitute the position such as the factor VIII(a) light chain associated with FIXa and FX on ACE910-tenase assembly defecting factor VIII. Disclosures Minami: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: The hemostatic effect of bypassing agents such as recombinant (r) factor (F)VIIa and activated prothrombin complex concentrates (aPCC) for hemophilia A with inhibitors (HA-inh) is not always stable (Berntope, Haemophilia 2009). The mechanism(s) of its instability remain unclear, however. We have recently reported the HA-inh case showing the attenuated responsiveness to aPCC (Ogiwara, Int J Hematol. 2014). Some groups reported the hemostatic effects of the complex concentrates of FVIIa and FX (Shirahata, Haemophilia 2012) in HA-inh, suggesting that FX would play the key role in the hemostatic effect by FVIIa. ACE910, a humanized bispecific antibody to FIXa and FX mimicking the functions of FVIIIa, exerting FXase activities without FVIII(a) (Kitazawa, Nature Medicine 2012). In this study, we attempted to elucidate the dependency on FX of the FVIIa- and/or ACE910-driven coagulation. Firstly, the global hemostatic potentials in the whole blood samples obtained from the four HA-inh cases (Case 1, 2, 3 and 4) under perioperative hemostatic treatment with the intermittent administration of rFVIIa every 2-3hr were evaluated by Ca2+-triggered viscoelastometric assay with ROTEM. The first infusion of rFVIIa shortened CT (from 5,087 ± 1,261 to 1,157 ± 208 sec) and increased MCF (from 17 ± 8.7 to 58.8 ± 1.3 mm) in each case. Additional rFVIIa after the 7th administration in Case 1, the 13th in Case 2 and the 12th in Case 3 little affected CT and MCF as well as clinical symptom, indicative of poor responsiveness, while Case 4 showed the improvement of the parameters even after the frequent infusion of rFVIIa, identified as a responsive case. Thrombin generation (TG) triggered by TF (1pM) or TF (1pM) together with ellagic acid (0.3μM) was evaluated in the plasma from the cases with poor response. Peak thrombin (PeakTh) was little changed between pre- and post-additional infusion of rFVIIa in the cases with poor response, similar to the pattern of ROTEM. The level of FX antigen measured by an ELISA in the plasma was 90.5 ± 9.6 nM, showing 67% of normal control (~140 nM), of little difference among the four cases at the first administration of rFVIIa, while that in Case 1, 2 or 3 at the 7th, 13th or 12th administration, respectively, decreased to 39.1 ± 7.0 nM, equivalent to ~45% of that (86.8 ± 12.9 nM) kept in the responsive Case 4. Addition of FX (300nM) in the plasma of poor response to rFVIIa ex vivo increased PeakTh to ~80% of normal control, suggesting that FVIIa-driven hemostatic effect would be dependent upon FX. Furthermore, to investigate the FX-dependency of FVIIa- and ACE910-driven coagulation, TG in the reconstituted HA-inh model plasmas consisting of FX-deficient plasma in which FVIII was inactivated by an anti-FVIII polyclonal antibody (10BU/ml) with/without rFVIIa (50 and 150 nM) or ACE910 (10, 30 and 60 μg/ml) was evaluated in the presence of various concentrations of FX (f.c. 0 - 300 nM). The control experiment without rFVIIa or ACE910 showed the FX dose-dependent increase of PeakTh. In the plasmas with FX ranged from 50 to 300nM, PeakTh improved to almost normal level by rFVIIa as well as ACE910. Of note, with the lower concentration of FX (10-20 nM), PeakTh improved to almost normal level in the presence of ACE910, increased by 38 ± 2.4%, 45 ± 1.7% and 48 ± 0.8% compared to those in its absence, respectively, in an ACE910 dose-dependent manner, whilst the presence of rFVIIa little affected TG compared to those in its absence. Taken together, ACE910 could exert its hemostatic effect with the lower amount of FX than that required for the rFVIIa-driven coagulation. Disclosures Yada: Chugai Pharmaceutical Co., ltd: Research Funding. Nogami:Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees. Kitazawa:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Shima:Biogen: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Kaketsuken: Honoraria.

Description: Background Factor VIII (FVIII) is an essential factor for coagulation system in the intrinsic pathway. Due to the short survival of FVIII in the plasma circulation, it requires von Willebrand factor (VWF) as a carrier protein to maintain the optimal level for hemostasis. VWF also plays an important role in primary hemostasis by bridging platelets to exposed subendothelial collagens, especially under high shear flow environment. Since VWF carries FVIII, it is conceivable that VWF takes FVIII to the sites of vascular injury. However, the role of FVIII at the local sites under flow conditions is not fully understood despite of the fact that increased level of FVIII is associated with the risk of venous thrombosis and the deficiency of FVIII is the pathology of the bleeding disorder, hemophilia A. The treatment of hemophilia A largely depends on the infusion of FVIII concentrates, which is often complicated by the development of the inhibitor. Recently, bispecific antibody（ACE910）that mimics the role of FVIIIa by recognizing FIXa and FX has been developed and is currently under clinical trial. This antibody theoretically works regardless of the presence of devastating inhibitors against FVIII. Furthermore, it could also improve the clinical outcome of the other bleeding disorders, such as von Willebrand disease (VWD). Aim To analyze the role of FVIII and VWF, and impact of ACE910 at the sites of vascular injury under various shear conditions, we have developed the flow-mediated thrombosis model using flow chamber system. Method Whole blood obtained from healthy donors, hemophilia A and VWD patients were perfused into the collagen coated flow chamber under high (2,500s-1) or low shear (50s-1) flow conditions with/without FVIII concentrate, FVIII/VWF concentrate and ACE910. Formed thrombus was fixed and immunostaining was performed with phalloidin (Platelet), anti-FVIII antibody (FVIII) and anti-thrombin antibody (Thrombin). For the detection of ACE910, anti-human IgG or anti-ACE antibody (rAQ8 or rAJ540) were used. Size of thrombi and distribution of platelet, FVIII, thrombin and ACE910 were analyzed. Result 1) Under high shear flow, thrombus formation of VWD blood was significantly impaired while blood from Hemophilia A demonstrated nearly normal thrombus formation. Addition of FVIII/VWF but not FVIII concentrate to the blood of these patients rescued the impaired thrombus formation. ACE910 enhanced the thrombus formation of blood from both VWD and hemophilia A. Under low shear flow, blood from both hemophilia A and VWD demonstrated decreased thrombus formation. FVIII, FVIII/VWF concentrates and ACE910 improved the size of thrombus. 2) Localization of FVIII was evaluated with thrombin as a marker for the activation of coagulation. Platelets and thrombin demonstrated complete co-localization and intensity of thrombin staining was associated with thrombus size. VWF localized mainly outer layer of thrombus and FVIII localized in and around thrombus. At high shear condition, FVIII and VWF mostly existed with platelets. By contrast, FVIII and VWF demonstrated less co-localization with platelets under low shear condition. ACE910 demonstrated similar tendency to FVIII localization although ACE910 did not appear around thrombus. Conclusion We have developed the flow chamber system to evaluate the extent of thrombogenesis under various shear environment. VWF showed dominant role under high shear conditions while FVIII plays a key role under low shear conditions. FVIII, VWF and ACE910 demonstrated distinct localization. Interestingly, the distribution of FVIII was broader than VWF and platelet. FVIII localized to platelets presumably prior to its activation and contributed for the subsequent thrombin generation at local sites. Finally, ACE910 demonstrated consistent enhancement of thrombus formation of blood from both hemophilia A and VWD and, therefore, is prompted for the treatment of these bleeding disorders. Disclosures Shida: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Minami:Chugai Pharmaceutical Co., Ltd.: Research Funding. Yaoi:Chugai Pharmaceutical Co., Ltd.: Research Funding. Matsumoto:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Acquired hemophilia A (AHA) is a rare bleeding disorder in which autoantibodies (autoAbs) against coagulation factor (F)VIII impair the coagulation system. Anti-FVIII autoAbs developed in AHA are polyclonal and the majority of these autoAbs bind to the A2, A3, and/or C2 domains in FVIII. Different inhibitory actions of anti-FVIII autoAbs, depending on their epitopes, are well-known. We have recently developed a humanized anti-FIXa/X bispecific antibody, ACE910, which mimics the cofactor function of FVIIIa even in the presence of anti-FVIII alloantibodies. In this study, we examined whether ACE910 improved the lower coagulation function in AHA patients using the comprehensive coagulation assays. Two representative experiments were performed as follows; 1) In vitro effects of ACE910 on normal pooled plasmas (PNPs) mixed with various anti-FVIII monoclonal Abs (mAbs) as the reconstituted AHA-models. For this experiments, several anti-FVIII mAbs; VIII-9222 (epitope: anti-a1), VIII-2236 (anti-A2), VIII-3776 (anti-A3C1), ESH4 (anti-C2) were prepared. 2) Ex vivo effects of ACE910 on plasmas obtained from nine patients with AHA, whose regions of Abs were A1, A2 and light chain for four patients, A2 and light chain for three patients, A2 for one patient and light chain for one patient. The coagulation functions in obtained samples were evaluated using the comprehensive coagulation assays, clot waveform analysis (CWA) and thrombin generation assays (TGA). In CWA, the minimum value of the first derivative (min1) and the second derivative (min2) were calculated as an index of the maximum acceleration of the reaction and the maximum velocity of coagulation, respectively. In TGA, calibrated automated thrombin generation assays derived the standard parameters; peak thrombin, lagtime, and time to peak. Total thrombin generation at intervals from the beginning to peak level was quantified. These values (nmol/l) were divided by the sample times (min, time to peak - lagtime), and represented the mean velocity of thrombin generation until the reach to peak level, expressed as mean velocity to peak thrombin (MV-peak thrombin). We defined the attenuation of inhibitory effect (% of AIE) represented that ACE910 attenuated the parameters inhibited by mAbs in artificial AHA or by allo-Abs in AHA patients, respectively. Figure 1 represents the % of AIE on thrombin peak in TGA. PNPs were incubated with various anti-FVIII mAbs (100-500 mcg/ml) at 37 degrees Celsius for 2 hrs, and FVIII activities in these samples were decreased to 2.0-43%. These reactant mixtures were reacted with ACE910 (0-20 mcg/ml) for a further 2 hr-incubation. ACE910 improved the parameters of CWA (|min1|; 40-62%, and |min2|; 49-74% of AIE) and of TGA (thrombin peak; 46-72%, and MV-peak thrombin; 51-70% of AIE, respectively) in all PNPs with mAbs. These data indicated that ACE910 improved the coagulant activity in AHA patients. Therefore, we determined the efficacy of ACE910 for nine AHA patients ex vivo. FVIII activities and FVIII inhibitors were

Description: Abstract 1126 Exogenous factor VIII (FVIII) is used to reduce bleeding complications in patients with severe hemophilia A. However, there are two drawbacks of current routine prophylaxis by FVIII. One is the requirement of frequent intravenous administration due to its short half-life and low subcutaneous bioavailability of FVIII. Second is the development of anti-FVIII antibodies (inhibitors) in approximately 30% of the severe patients which deprives the patients from routine prophylaxis by FVIII. To overcome these drawbacks, bispecific IgG antibody against activated factor IX (FIXa) and factor X (FX), which mimics the cofactor function of FVIII by placing these two factors into spatially appropriate positions, was screened from approximately 40,000 bispecific antibodies recognizing FIXa by the one arm and FX by the other arm. The therapeutic potential of the bispecific antibody identified from the screening was marginal due to insufficient FVIII-mimetic activity and poor pharmacokinetics, and moreover, large scale purification of recombinant bispecific IgG antibody was challenging. Therefore, the lead bispecific antibody, after humanization, was subjected to multidimensional optimization process in order to improve both the therapeutic potential and the manufacturability of the bispecific antibody. FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulty of manufacturing bispecific antibody was overcome by identifying common light chain for anti-FIXa and FX heavy chain through framework/complementarity determining region shuffling, and by isoelectric point engineering of the two heavy chain variable regions to facilitate ion exchange chromatography purification of the bispecific antibody. Engineering to overcome low solubility and deamidation was also performed to enable stable high concentration liquid formulation for clinical use. ACE910, multidimensionally optimized bispecific antibody, exhibited potent FVIII-mimetic activity in human FVIII deficient plasma (more than 10% of FVIII activity at 300 nM in thrombin generation assay), and half-life of approximately 3 weeks with high subcutaneous bioavailability in cynomolgus monkey, enabling effective prophylaxis by subcutaneous administration with long dosing interval. In silico immunogenicity prediction analysis suggested that ACE910 was minimally immunogenic in human, in contrast to high immunogenicity of FVIII in human. Importantly, the activity of ACE910 was not affected by the presence of inhibitors, while polyclonal anti-ACE910 antibody did not inhibit FVIII activity, allowing the use of ACE910 without considering the development or presence of inhibitors. Furthermore, ACE910 could be purified in a large scale manufacturing, and formulated into patient-friendly subcutaneously injectable liquid formulation for clinical use. We believe that ACE910, with its multidimensionally optimized profile, would significantly improve the quality of life of hemophilia A patients by reducing not only bleeding but also the burden on the patients themselves, their parents, and all medical staff. Disclosures: Igawa: Chugai Pharmaceutical Co.,Ltd: Employment. Sampei:Chugai Pharmaceutical Co., Ltd.: Employment. Soeda:Chugai Pharmaceutical Co.,Ltd: Employment. Okuyama-Nishida:Chugai Pharmaceutical Co., Ltd.: Employment. Moriyama:Chugai Pharmaceutical Co.,Ltd: Employment. Wakabayashi:Chugai Pharmaceutical Co.,Ltd: Employment. Tanaka:Chugai Pharmaceutical Co.,Ltd: Employment. Muto:Chugai Pharmaceutical Co., Ltd.: Employment. Kojima:Chugai Pharmaceutical Co.,Ltd: Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yoshihashi:Chugai Pharmaceutical Co.,Ltd: Employment. Harada:Chugai Pharmaceutical Co.,Ltd: Employment. Funaki:Chugai Pharmaceutical Co.,Ltd: Employment. Haraya:Chugai Pharmaceutical Co.,Ltd: Employment. Tatsuhiko:Chugai Pharmaceutical Co.,Ltd: Employment. Suzuki:Chugai Pharmaceutical Co.,Ltd: Employment. Esaki:Chugai Pharmaceutical Co.,Ltd: Employment. Nabuchi:Chugai Pharmaceutical Co.,Ltd: Employment. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment.

Description: Key Points A long-term acquired hemophilia A model expressing spontaneous joint bleeds and other bleeds was newly established in nonhuman primates. Weekly SC dose of the anti-FIXa/X bispecific antibody ACE910 prevented joint bleeds and other bleeds in the primate hemophilia A model.

Description: Modulating the complement system is a promising approach in drug discovery for overcoming uncontrolled complement activation, which is known to cause various disorders. While these disorders can be effectively treated by inhibiting complement C5 with a therapeutic antibody, the high concentration of C5 in plasma requires a huge dosage and frequent intravenous administration of the antibody. We have generated SKY59, an engineered humanized anti-C5 monoclonal antibody that shows long-lasting neutralization of C5 in cynomolgus monkeys, which enables subcutaneous delivery or less frequent administration. We found that, although the effective duration of the antibody was prolonged by engineering a pH-dependent C5 binding, it was not sufficient to achieve subcutaneous delivery with less frequent administration. Here, we further improved the effectiveness of the pH-dependent anti-C5 antibody by applying a novel approach: surface charge engineering. Intravenous injection of 40 mg/kg CFA0322, a high affinity anti-C5 monoclonal antibody that is not pH-dependent, caused about 3-fold increase of total C5 concentration in the plasma of cynomolgus monkeys. This is because the C5-antibody immune complexes (ICs) have slower clearance than C5 alone. The increase in total C5 concentration then requires an even higher dosage of the antibody to neutralize the increased amount of C5. To reduce the required dosage of anti-C5 antibody, pH-dependent antibodies were identified from rabbit immunization. A humanized lead antibody, 305LO1, showed a pH-dependent binding property and reduced the plasma total C5 concentration compared to CFA0322. However, we still observed an increase in total C5 plasma concentration over the baseline. We assumed that this was due to a slow uptake rate of the C5-antibody ICs into the cell. We hypothesized that the surface charges of the ICs partially contributed to the slow uptake rate. Surface-charge engineered antibodies were generated by engineering both the variable and the constant regions. Both approaches successfully reduced the increase of plasma C5 in cynomolgus monkey and the combination of the two completely suppressed the accumulation. Interestingly, surface-charge engineering did not affect the pharmacokinetics of the antibody. The effect of surface-charge engineering on C5 reduction correlated clearly with the retention time of the C5-antibody ICs in cation exchange chromatography, indicating the contribution of positive surface charges of the ICs. After further engineering to optimize various properties such as immunogenicity and physicochemical properties, SKY59 was successfully generated. Our surface-charge engineered pH-dependent antibody suppressed the increase of total C5 concentration in vivo by accelerating the rate of IC uptake into cells, C5 release from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, SKY59 can be expected to provide significant benefits for patients with complement-mediated disorders. Disclosures Kitazawa: Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties: Patents related to emicizumab.

Description: Abstract 42 Background: Hemophilia A is treated by intravenous replacement therapy with factor VIII (FVIII), either on demand to resolve bleeding or as a prophylactic to prevent bleeding. Recently, routine prophylactic treatment is recommended to effectively prevent bleeding and to reduce bleeding-related chronic joint damage. However, the need for frequent intravenous injections of FVIII negatively affects patients' quality of life and their adherence to the routine prophylactic regimen. More importantly, approximately 30% of severe hemophilia A patients develop inhibitory antibodies toward the injected FVIII, rendering the replacement therapy ineffective. To overcome these drawbacks, we generated a bispecific antibody (termed ACE910) against activated factor IX (FIXa) and factor X (FX), which mimics the cofactor function of FVIII. Objectives: The aims of the present study were to examine the FVIII-mimetic cofactor activity of ACE910 in vitro and its hemostatic activity in vivo. Methods: The FVIII-mimetic cofactor activity of ACE910 was evaluated by a thrombin generation assay in human FVIII-deficient plasma as well as by an enzymatic assay using purified coagulation factors. For in vivo studies, an acquired hemophilia A model was established in cynomolgus monkeys by a single intravenous injection of mouse monoclonal anti-FVIII neutralizing antibody, which was cross-reactive to cynomolgus monkey FVIII but not to porcine FVIII. After artificial bleeding had been induced, ACE910 or porcine FVIII was intravenously administered in a single dose or in twice-daily repeated doses, respectively. Bleeding symptoms, including anemia and skin bruising, were monitored for three days. A pharmacokinetic study of ACE910 was also performed with a single intravenous or subcutaneous administration to cynomolgus monkeys. Results: ACE910 concentration-dependently showed FVIII-mimetic cofactor activity in the enzymatic assay and improved thrombin generation parameters in human FVIII-deficient plasma. Intravenous administration of ACE910 (a single dose of 3 mg/kg) significantly reduced the bleeding symptoms in the acquired hemophilia A model of a non-human primate. This hemostatic effect was comparable to twice-daily intravenous administration of porcine FVIII (repeated doses of 10 U/kg). The half-life of ACE910 was approximately three weeks for both single intravenous and subcutaneous administrations. The subcutaneous bioavailability of ACE910 was nearly 100%. Conclusion: The bispecific antibody against FIXa and FX, ACE910, exerted FVIII-mimetic cofactor activity in vitro. Furthermore, a single dose of ACE910 demonstrated hemostatic activity comparable to twice-daily repeated doses of 10 U/kg porcine FVIII in vivo. Moreover, ACE910 exhibited high subcutaneous bioavailability and approximately three-week half-life in a non-human primate. Our bispecific antibody against FIXa and FX is a subcutaneously injectable, long-acting agent that removes the need to consider the induction or presence of FVIII inhibitors and may establish a novel principle for the prophylactic treatment of hemophilia A patients. Disclosures: Muto: Chugai Pharmaceutical Co., Ltd.: Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yoshihasi:Chugai Pharmaceutical Co., Ltd.: Employment. Takeda:Chugai Pharmaceutical Co., Ltd.: Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Employment. Sampei:Chugai Pharmaceutical Co., Ltd.: Employment. Sakamoto:Chugai Pharmaceutical Co., Ltd.: Employment. Okuyama-Nishida:Chugai Pharmaceutical Co., Ltd.: Employment. Saito:Chugai Pharmaceutical Co., Ltd.: Employment. Kawabe:Chugai Pharmaceutical Co., Ltd.: Employment. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment.

Description: Background : Factor VIII (FVIII) is protected by binding to its carrier protein, von Willebrand factor (VWF) in the circulation. VWF contributes to hemostasis particularly under high shear flow condition by extending its multimeric configuration. By contrast, coagulation factors including FVIII are thought to play a dominant role under low shear. Since FVIII binds to VWF unlike other coagulation factors, FVIII may contribute to thrombus formation even under high shear conditions. FVIII has to be released from VWF and activated to exert its coagulation function. However, the role of interaction between FVIII and VWF on hemostasis under flow conditions needs to be explored in addition to the protective function of VWF. Aims: To analyze the mechanism and role of FVIII and VWF interaction on hemostasis under flow conditions. Methods: Whole blood samples were obtained from patients with type 2A von Willebrand disease (VWD). FVIII, VWF, FVIII/VWF (final concentration; f.c. 1U/mL), FVIII plus ESH8 (f.c. 1 µM), and emicizumab (ACE910; f.c. 100 µg/mL) were added to the blood followed by perfusion into the collagen-coated flow chamber under controlled high (2,500s-1) and low (50s-1) shear conditions. ESH8 is an anti-FVIII antibody that interrupts the release of FVIII from VWF, and emicizumab is a bispecific antibody mimicking activated FVIII (FVIIIa). The concentration of emicizumab was chosen, as it was the highest dose in clinical study. After the perfusion, formed thrombus was fixed and immunostaining was performed to visualize platelets, VWF, thrombin, and FVIII. Thrombi were observed by using confocal laser scanning microscopy and the obtained images were analyzed by Image Pro Premier 3D. Initial thrombus formation was measured as surface coverage (SC) and thrombus development was measured as thrombus volume (TV). Results: SC and TV were impaired in VWD particularly under high shear (high: SC 8.3%, TV 3.9x103μm3, low: SC 2.7%, TV 2.9x103μm3). Addition of FVIII/VWF improved thrombus formation to normal level under both shear conditions (high: SC 37.3%, TV 2.4x104μm3, low SC 4.3%, TV 4.1x103μm3). Addition of VWF improved SC and TV under both shear conditions (high: 40.9%, 1.7x104μm3, low: SC 3.0%, TV 3.3x103μm3), suggesting that the VWF function was crucial under both shear. By contrast, addition of FVIII alone did not improve SC and TV under both shears (high: SC 9.9%, TV 5.0x103μm3, low: SC 2.4%, TV 3.1x103μm3). Since FVIII/VWF had a greater effect on TV than VWF alone, FVIII enhanced thrombus development under high shears, however, this effect required the presence of VWF. FVIII immunostaining demonstrated the binding of FVIII on platelet surface in the FVIII-added experiment and, therefore, FVIII binding alone was not sufficient to initiate coagulation. Since FVIII may need to be activated on the platelet surface in a timely fashion, it was speculated that the optimal delivery from VWF and activation of FVIII on platelets was required for hemostasis. To analyze the role of FVIII and VWF interaction on hemostasis under flow condition, ESH8 was used to modify FVIII/VWF binding by blocking FVIII release. Treatment of FVIII/VWF with ESH8 did not change SC but slightly impaired TV under high shear (high: SC 41.8%, TV 1.8 x104μm3). Thus, the presence of FVIII at thrombus was not enough and the release of FVIII from VWF and presumably activation of FVIII on the platelet surface might be essential for thrombus formation under high shear. Under low shear, both SC and TV were interrupted by ESH8 (SC 2.6%, TV 3.1x103μm3), suggesting the important role of FVIII and coagulation under low shear. Lastly, to analyze the impact of activated FVIII on thrombus formation under flow conditions, emicizumab was evaluated in this system. In contrast to FVIII, emicizumab alone improved thrombus formation (high: SC 26.0%, TV 1.1 x104μm3, low: SC 3.5%, TV 4.2 x103μm3). The rate of improvement in SC and TV was better under low shear than high shear, which implicated the coagulative role of emicizumab. Even under high shear, however, emicizumab enhanced thrombus formation. Therefore, the role of emicizumab in substituting for the activated form of FVIII might be able to rapidly initiate coagulation even under high shear. Conclusion: VWF delivers and releases FVIII in a timely fashion on platelet surface. FVIII has to become active on platelets for hemostasis and FVIIIa can accelerate thrombus formation even under high shear condition. Disclosures Yaoi: Chugai Pharmaceutical Co., Ltd.: Research Funding. Shida:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kitazawa:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties; Sysmex Corporation: Patents & Royalties. Hattori:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties. Shima:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Nogami:F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.