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High Affinity Binding of a MUC1 Specific Antibody to a Glycopeptide Mediated by Conformational Selection
Author(s) -
Brooks Cory L,
Movahedin Reza,
Brooks Teresa M,
Supekar Nitin,
Boons GeertJan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1097.3
Subject(s) - glycopeptide , epitope , glycosylation , antibody , peptide , glycan , chemistry , muc1 , glycoprotein , biochemistry , antigen , biology , mucin , immunology , antibiotics
MUC1 is a transmembrane glycoprotein and is a target for antibody‐mediated immunotherapy. In cancer cells, MUC1 is upregulated, and undergoes abnormal glycosylation resulting in truncated carbohydrate chains being added to the protein. The truncated glycosylation exposes cryptic peptide epitopes that can be recognized by antibodies. Since these immunogenic regions are cancer specific, they represent ideal targets for therapeutic antibodies. The peptide sequence, and the cancer specific truncated glycans are both available for antibody recognition. There is significant interest in determining which is the more suitable target for antibody immunotherapy. We investigated the role of tumor specific glycosylation in antigen recognition by the therapeutic antibody AR20.5. Using microscale thermophoresis we explored the affinity of AR20.5 to cancer specific MUC1 glycopeptide and peptide. The antibody bound to the glycopeptide with an order of magnitude stronger affinity than against the naked peptide. Given these results we postulated that A20.5 must specifically bind the carbohydrate as well as the peptide. Using X‐ray crystallography we examined this hypothesis by determining the structure of AR20.5 in complex with both peptide and glycopeptide. Surprisingly, the structure revealed that the carbohydrate did not form any specific contacts with the antibody. The high affinity of AR20.5 for the glycopeptide and the lack of specific binding contacts supports a hypothesis that glycosylation of MUC1 stabilizes an extended bioactive conformation of the peptide that is recognized by the antibody. High affinity binding of AR20.5 to the MUC1 glycopeptide is not driven by specific antibody‐antigen contacts, but rather glycosylation alters the conformational equilibrium of the antigen which allows the antibody to select the correct coformation. This study demonstrates a novel mechanism of antibody‐antigen interaction and also suggests that glycosylation of MUC1 is important for the generation of high affinity therapeutic antibodies. Support or Funding Information This work was supported through start‐up funds from the College of Science and Math at CSUF, and by a research grant from Quest Pharmatech Inc. to CLB. MR was supported by rGrants from ASI at CSUF and FSSRA awards from the College of Science and Math at CSUF.