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ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required.

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.

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.