Factor VIII Mimetic Antibody: (1) Establishment and Characterization of Anti-factor IX/Anti-factor X Bispecific Antibodies

Abstract number: OR160

Saito¹ H, Kojima¹ T, Miyazaki¹ T, Soeda¹ T, Kitazawa¹ T, Suzuki¹ T, Yoshihashi¹ K, Adachi¹ H, Nezu¹ J, Shima² M, Yoshioka² A, Hattori¹ K

¹¹Chugai Pharmaceutical Co., Ltd., Gotemba, Japan ¹¹Chugai Pharmaceutical Co., Ltd., Gotemba, Japan ²²Nara Medical University, Kashihara, Japan

Two major problems for hemophilia A treatment with factor VIII (FVIII) concentrates are development of FVIII inhibitors and inconvenience due to frequent intravenous injections. Antibodies mimicking FVIII activity could be an ideal therapeutics for hemophilia A, because such antibodies possibly have a long-lasting therapeutic effect even in the patients with inhibitors. As the cofactor function of FVIIIa to promote activation of factor X (FX) is hypothesized to fix factor IXa (FIXa) and FX in an appropriate position and angle, we expected bispecific antibodies which recognize FIX and FX to mimic the action of FVIIIa. For bispecific antibody expression, genes coding the VH and VL regions of anti-FIX antibodies and anti-FX antibodies derived from hybridomas were inserted into mammalian vectors designed for bispecific human IgG4. Antibodies were screened for activity to promote FX activation by FIXa in a chromogenic assay and to shorten the modified APTT in FVIII deficient plasma. Seventeen bispecific antibodies accelerated the FX activation in the absence of FVIII, which were shown to shorten the clotting time in FVIII deficient plasma. The most potent antibody, XB12/SB04 (anti-FIX/anti-FX), shortened the modified APTT in FVIII deficient plasma from 115.5 seconds to 61.3 seconds at 10 mg/mL (normal plasma: 34.3 seconds). XB12/SB04 demonstrated no competition with FVIII. The epitope of most anti-FX antibodies including SB04 were found to exist in activation peptide, which is cleaved from FX upon activation. It was suggested that the recognition of the activation peptide contributes to the coagulant activity by the release of FXa to form prothrombinase. These results demonstrate that the cofactor function of FVIII can be mimicked by bispecific antibodies to FIX and FX and that XB12/SB04 could be a long-acting therapeutics for hemophilia A, which is effective even in patients with inhibitors.