title: Three-dimensional structures of a humanized anti-IFN-gamma Fab (HuZAF) in two crystal forms.
abstract: Three-dimensional structures were determined for two crystal forms (orthorhombic P2(1)2(1)2(1) 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 gamma (IFN-gamma). 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-gamma 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-gamma 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-gamma antigen. Thus, there are currently three slightly different structures of the HuZAF Fab.
authors.affiliation: Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, OK 74078-2007, USA.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Bourne
authors.forename: Simon S
authors.initials: SS
authors.lastname: Terzyan
authors.forename: Gwendolyn
authors.initials: G
authors.lastname: Cloud
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Landolfi
authors.forename: Maximiliano
authors.initials: M
authors.lastname: Vásquez
authors.forename: Allen B
authors.initials: AB
authors.lastname: Edmundson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007371:Interferon-gamma / Q000737:chemistry* / Q000276:immunology; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction; D014965:X-Rays
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D008024:Ligands; D007371:Interferon-gamma