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Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain.
- Source :
-
Journal of molecular recognition : JMR [J Mol Recognit] 2012 Mar; Vol. 25 (3), pp. 125-35. - Publication Year :
- 2012
-
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.<br /> (Copyright © 2012 John Wiley & Sons, Ltd.)
- Subjects :
- Amino Acid Sequence
Antibodies, Monoclonal pharmacology
Antibodies, Neutralizing pharmacology
Antibody Affinity
Binding Sites, Antibody
Cells, Cultured
Cysteine chemistry
Cytokines chemistry
Cytokines immunology
Epitopes chemistry
Epitopes immunology
Humans
Molecular Dynamics Simulation
Molecular Sequence Data
Peptide Fragments chemistry
Peptide Fragments isolation & purification
Peptide Library
Peptide Mapping
Phosphorylation
Protein Binding
Protein Stability
Protein Structure, Secondary
STAT3 Transcription Factor metabolism
Solubility
Transition Temperature
Antibodies, Monoclonal chemistry
Antibodies, Neutralizing chemistry
Complementarity Determining Regions chemistry
Disulfides chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1099-1352
- Volume :
- 25
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Journal of molecular recognition : JMR
- Publication Type :
- Academic Journal
- Accession number :
- 22407976
- Full Text :
- https://doi.org/10.1002/jmr.1168