title: Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain.
abstract: Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4 nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.
authors.affiliation: Centocor R&D Inc, Biologics Research, Radnor, PA 19087, USA.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Almagro
authors.forename: Gopalan
authors.initials: G
authors.lastname: Raghunathan
authors.forename: Eric
authors.initials: E
authors.lastname: Beil
authors.forename: Dariusz J
authors.initials: DJ
authors.lastname: Janecki
authors.forename: Qiang
authors.initials: Q
authors.lastname: Chen
authors.forename: Thai
authors.initials: T
authors.lastname: Dinh
authors.forename: Ann
authors.initials: A
authors.lastname: LaCombe
authors.forename: Judy
authors.initials: J
authors.lastname: Connor
authors.forename: Mark
authors.initials: M
authors.lastname: Ware
authors.forename: Paul H
authors.initials: PH
authors.lastname: Kim
authors.forename: Ronald V
authors.initials: RV
authors.lastname: Swanson
authors.forename: Johan
authors.initials: J
authors.lastname: Fransson
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000494:pharmacology; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D002478:Cells, Cultured; D022801:Complementarity Determining Regions / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D016207:Cytokines / Q000737:chemistry / Q000276:immunology; D004220:Disulfides / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D019151:Peptide Library; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D050796:STAT3 Transcription Factor / Q000378:metabolism; D012995:Solubility; D044366:Transition Temperature
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D022801:Complementarity Determining Regions; D016207:Cytokines; D004220:Disulfides; D000939:Epitopes; D010446:Peptide Fragments; D019151:Peptide Library; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D003545:Cysteine