Three-dimensional structures of a humanized anti-IFN-gamma Fab (HuZAF) in two crystal forms.
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.
Bourne
Philip C
PC
Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, OK 74078-2007, USA.
Terzyan
Simon S
SS
Cloud
Gwendolyn
G
Landolfi
Nicholas F
NF
Vásquez
Maximiliano
M
Edmundson
Allen B
AB
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
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D008024:Ligands; D007371:Interferon-gamma