title: An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity.
abstract: The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system. The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect. However, this murine IgM/kappa antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure-function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis. We describe here the affinity maturation of a bacterially produced functional IN-1 F(ab) fragment via protein engineering. A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments. After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 F(ab) fragment was obtained, carrying five side chain substitutions within CDR-L3. Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 microM. The affinity of the unmutated IN-1 F(ab) fragment was 8-fold lower. The engineered F(ab) fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections. Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture. This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered F(ab) fragment as a reagent for promoting axonal regeneration in vivo.
authors.affiliation: Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Fiedler
authors.forename: C
authors.initials: C
authors.lastname: Horn
authors.forename: C
authors.initials: C
authors.lastname: Bandtlow
authors.forename: M E
authors.initials: ME
authors.lastname: Schwab
authors.forename: A
authors.initials: A
authors.lastname: Skerra
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D000818:Animals; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D007120:Immunochemistry; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D009185:Myelin Proteins / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D000070798:Nogo Proteins; D010455:Peptides / Q000378:metabolism; D015202:Protein Engineering; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D007140:Immunoglobulin Fab Fragments; D009185:Myelin Proteins; D000070798:Nogo Proteins; D010455:Peptides; C000603197:Rtn4 protein, rat