title: Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin.
abstract: Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.
authors.affiliation: Pepscan Therapeutics, Zuidersluisweg 2, 8203RC Lelystad, The Netherlands. j.back@pepscan.com
authors.forename: J W
authors.initials: JW
authors.lastname: Back
authors.forename: C
authors.initials: C
authors.lastname: Frisch
authors.forename: K
authors.initials: K
authors.lastname: Van Pee
authors.forename: V
authors.initials: V
authors.lastname: Boschert
authors.forename: R
authors.initials: R
authors.lastname: van Vught
authors.forename: W
authors.initials: W
authors.lastname: Puijk
authors.forename: T D
authors.initials: TD
authors.lastname: Mueller
authors.forename: A
authors.initials: A
authors.lastname: Knappik
authors.forename: P
authors.initials: P
authors.lastname: Timmerman
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D003553:Cystine / Q000737:chemistry; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D020349:Surface Plasmon Resonance; D060449:Wnt Signaling Pathway / Q000187:drug effects*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D011506:Proteins; D011994:Recombinant Proteins; C480733:SOSTDC1 protein, human; D003553:Cystine