title: A novel and conserved pocket of human kappa-Fab fragments: design, synthesis, and verification of directed affinity ligands.
abstract: Antibodies of type IgG may be divided into two classes, called lambda or kappa, depending on the type of light chain. We have identified a conserved pocket between the two domains CH1 and CL of human IgG kappa-Fab, which is not present in the lambda type. This pocket was used as a target docking site with the purpose of exploring the possibilities of designing affinity ligands that could function as such even after immobilization to gel. The idea of the design arose mainly from the results of the saturated transfer difference (STD-NMR) screening of 46 compounds identified by means of virtual docking of 60 K diverse compounds from the Available Chemicals Directory (ACD). Surface plasmon resonance (SPR) was used as an alternative method to monitor binding in solution. A total of 24 compounds belonging to a directed library were designed, synthesized, and screened in solution. They consist essentially of an amino acid condensed to a N,N'-methylated phenyl urea. STD-NMR results suggest that a small hydrophobic side chain in the condensed amino acid promotes binding, whereas a hydroxyl-group-containing side chain implies absence of STD-NMR signals. Three compounds of the directed library were immobilized and evaluated as chromatographic probes. In one case, using D-Pro as the condensed amino acid, columns packed with ligand-coupled Sepharose (Amersham Biosciences) retained two different monoclonal samples of kappa-Fab fragments with different variable regions, whereas a sample of monoclonal lambda-Fab fragments was not retained under similar chromatographic conditions.
authors.affiliation: Amersham Biosciences R&D, Björkgatan 30, S-751 84 Uppsala, Sweden.
authors.forename: Enrique
authors.initials: E
authors.lastname: Carredano
authors.forename: Herbert
authors.initials: H
authors.lastname: Baumann
authors.forename: Anna
authors.initials: A
authors.lastname: Grönberg
authors.forename: Nils
authors.initials: N
authors.lastname: Norrman
authors.forename: Gunnar
authors.initials: G
authors.lastname: Glad
authors.forename: Jinyu
authors.initials: J
authors.lastname: Zou
authors.forename: Oguz
authors.initials: O
authors.lastname: Ersoy
authors.forename: Elles
authors.initials: E
authors.lastname: Steensma
authors.forename: Andreas
authors.initials: A
authors.lastname: Axén
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D017124:Conserved Sequence; D015195:Drug Design; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D019151:Peptide Library; D011485:Protein Binding; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D019151:Peptide Library