Three-dimensional structures of a humanized anti-IFN-γ Fab (HuZAF) in two crystal forms

Three‐dimensional structures were determined for two crystal forms (orthorhombic P212121 and monoclinic C2) of the Fab from the humanized version of a murine monoclonal antibody (AF2) that possesses binding and potent neutralizing activity against human interferon γ (IFN‐γ). This humanized antibody (HuZAF; USAN name fontolizumab) is currently in phase II clinical trials for the treatment of Crohn's disease. HuZAF exhibits binding and IFN‐γ neutralizing capacities that closely approximate those of the original antibody. It is shown that HuZAF, whose VH domain was designed using a best‐sequence‐fit approach, is closer structurally to its mouse precursor than is a version whose VH was constructed using a human sequence with lower homology to the original mouse sequence. This work thus offers direct structural evidence in support of the best‐sequence‐fit approach and adds to previous results of biological and biochemical evaluations of distinctly engineered antibodies that also favored the use of a best‐sequence‐fit strategy. A second crystal type appeared during attempts to crystallize the Fab–IFN‐γ complex. The antibody–antigen complex that existed in solution dissociated in the crystallization mixture. A conformationally altered but unliganded HuZAF protein crystallized in a different space group (C2), with two Fab molecules in the asymmetric unit. In this crystal lattice, no space was available for accommodating the IFN‐γ antigen. Thus, there are currently three slightly different structures of the HuZAF Fab.