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Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers.

ABSTRACT: Although rabbit antibodies are widely used in research, no structures of rabbit antigen-binding fragments (Fab) have been reported. M204 is a rabbit monoclonal antibody that recognizes a generic epitope that is common to prefibrillar amyloid oligomers formed from many different amyloidogenic sequences. Amyloid oligomers are widely suspected to be a primary causative agent of pathogenesis in several age-related neurodegenerative diseases, such as Alzheimer's disease. The detailed structure of these amyloid oligomers is not known nor is the mechanism for the recognition of the generic epitope by conformation-dependent monoclonal antibodies.As a first approach to understanding the mechanism of conformation-dependent antibody recognition, we have crystallized the Fab of M204.We have determined the structure of the Fab of M204 at 1.54Å resolution. The crystal structure reveals details of the M204 antigen combining site and features unique to rabbit Fabs such as an interdomain disulfide bond on its light chain.Based on the structural features of the antigen-combining site of the M204, we rule out a "steric zipper" formation, as found in numerous amyloid fibril structures, as a mechanism of antibody-antigen recognition. The details of the first rabbit immunoglobulin Fab structure might also be useful for exploiting the potential of rabbit monoclonal antibodies for the development of humanized rabbit antibodies as therapeutic agents.

SUBMITTER: Arai H

PROVIDER: S-EPMC3973424 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers.

Arai Hiromi H Glabe Charles C Luecke Hartmut H

Biochimica et biophysica acta 20120824 12

<h4>Background</h4>Although rabbit antibodies are widely used in research, no structures of rabbit antigen-binding fragments (Fab) have been reported. M204 is a rabbit monoclonal antibody that recognizes a generic epitope that is common to prefibrillar amyloid oligomers formed from many different amyloidogenic sequences. Amyloid oligomers are widely suspected to be a primary causative agent of pathogenesis in several age-related neurodegenerative diseases, such as Alzheimer's disease. The detail ...[more]

PMID: 22940003

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Project description:IntroductionImmunotherapy targeting amyloid-β peptide is under active clinical investigation for treatment of Alzheimer's disease (AD). Among the hypotheses being investigated for impact on clinical outcome are the preferred epitope or conformation of amyloid-β to target for treatment, and the mechanism of action underlying immunotherapy. Bapineuzumab (humanized 3D6), a neo-epitope specific antibody recognizing amyloid-β1-5 with strong preference for an exposed Asp residue at the N-terminus of the peptide, has undergone advanced clinical testing for treatment of AD.MethodsTo gain further insight into the epitope conformation, we interrogated structural details of amino-terminal epitopes in amyloid-β using x-ray crystallography of 3D6Fab:amyloid-β complexes. Humanization of 3D6 was carried out using standard procedures integrating recombinant methods, sequence informatics, and homology modeling predictions to identify important mouse framework residues for retention in the finished humanized product.ResultsHere we report the crystal structure of a recombinant Fab fragment of 3D6 in complex with amyloid-β1-7 solved at 2.0 Å resolution. The N-terminus of amyloid-β is bound to 3D6 as a 310 helix. The amino-terminal Asp residue is buried deepest in the antibody binding pocket, with the Cβ atom of residue 6 visible at the entrance to the binding pocket near the surface of the antibody. We further evaluate homology model based predictions used to guide humanization of 3D6 to bapineuzumab, with actual structure of the Fab. The structure of the Fab:amyloid-β complex validates design of the humanized antibody, and confirms the amyloid-β epitope recognized by 3D6 as previously mapped by ELISA.ConclusionsThe conformation of amyloid-β antigen recognized by 3D6 is novel and distinct from other antibodies recognizing N-terminal epitopes. Our result provides the first report demonstrating structural conservation of antigen contact residues, and conformation of antigen recognized, between the parent murine antibody and its humanized version.

Dataset Information

Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers.

Publications

Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers.

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