title: Non-antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity.
abstract: While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG4 antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non-antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG4-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non-antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.
authors.affiliation: a Research Division; Chugai Pharmaceutical Co., Ltd ; Tokyo , Japan.
authors.forename: Zenjiro
authors.initials: Z
authors.lastname: Sampei
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Igawa
authors.forename: Tetsuhiro
authors.initials: T
authors.lastname: Soeda
authors.forename: Miho
authors.initials: M
authors.lastname: Funaki
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Yoshihashi
authors.forename: Takehisa
authors.initials: T
authors.lastname: Kitazawa
authors.forename: Atsushi
authors.initials: A
authors.lastname: Muto
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Kojima
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nakamura
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Hattori
mesh_terms: D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics; D040761:Biomimetic Materials / Q000737:chemistry*; D015949:Factor IXa / Q000737:chemistry*; D005169:Factor VIII; D005170:Factor X / Q000737:chemistry*; D006801:Humans
chemical_list: D018033:Antibodies, Bispecific; C078147:F8 protein, human; D005169:Factor VIII; D005170:Factor X; D015949:Factor IXa
keywords: FAE, Fab-arm exchange; FIX, coagulation factor IX; FIXa, activated coagulation factor IX; FVIII, coagulation factor VIII; FX, coagulation factor X; FXa, activated coagulation factor X; Fc glycosylation; IgG subclass; antibody engineering; bispecific antibody; constant region; disulfide bond; elbow angle; flexibility; hemophilia A; hinge