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: Philip C Bourne, Simon S Terzyan, Gwendolyn Cloud, Nicholas F Landolfi, Maximiliano Vásquez, Allen B Edmundson
• Journal: Acta crystallographica. Section D, Biological crystallography (Acta Crystallogr D Biol Crystallogr) Vol. 60 Issue Pt 10 Pg. 1761-9 (Oct 2004) ISSN: 0907-4449 [Print] United States
• PMID: 15388922 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antibodies, Monoclonal
• Immunoglobulin Fragments
• Ligands
• Interferon-gamma

Topics

• Amino Acid Sequence
• Animals
• Antibodies, Monoclonal (chemistry)
• Crystallization
• Crystallography, X-Ray
• Humans
• Immunoglobulin Fragments (chemistry)
• Interferon-gamma (chemistry, immunology)
• Ligands
• Mice
• Models, Molecular
• Molecular Sequence Data
• Protein Conformation
• Protein Structure, Tertiary
• Sequence Homology, Amino Acid
• X-Ray Diffraction
• X-Rays