Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain.

Almagro JC, Raghunathan G, Beil E, Janecki DJ, Chen Q, Dinh T, Lacombe A, Connor J, Ware M, Kim PH, Swanson RV, Fransson J.
Journal   J Mol Recognit.
Species  
Analytes Measured   STAT3
Matrix Tested   Assay buffer, cell lysates
Year   2012
Volume   25
Page Numbers   125-135
Application   Phosphoproteins
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.

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