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Title: Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [1] ;  [3] ;  [4] ;  [1] ;  [5] ;  [5] ;  [2] ;  [6] ;  [1]
  1. Univ. of Chicago, IL (United States). Dept. of Biochemistry and Molecular Biology
  2. Northwestern Univ., Evanston, IL (United States). Dept. of Chemistry. Dept. of Molecular Biosciences. Chemistry of Life Processes Inst.
  3. Univ. of Chicago, IL (United States). Dept. of Biochemistry and Molecular Biology; Wroclaw Univ. of Technology (Poland). Dept. of Biochemistry
  4. Univ. of Chicago, IL (United States). Dept. of Molecular Genetics and Cell Biology
  5. Univ. of North Carolina, Chapel Hill, NC (United States). School of Medicine. Dept. of Biochemistry and Biophysics
  6. Univ. of Chicago, IL (United States). Dept. of Biochemistry and Molecular Biology. Dept. of Molecular Genetics and Cell Biology
Publication Date:
OSTI Identifier:
1241064
Grant/Contract Number:
AC02-06CH11357; R21 DA025725; RC1 DA028779; GM067193; P30 CA014599
Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 113; Journal Issue: 8; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Univ. of Chicago, IL (United States)
Sponsoring Org:
USDOE Office of Science (SC); National Inst. of Health (NIH) (United States); W. M. Keck Foundation (United States); Chicago Biomedical Consortium (United States); Chicago Community Trust (United States)
Contributing Orgs:
Wroclaw Univ. of Technology (Poland); Northwestern Univ., Evanston, IL (United States); Univ. of North Carolina, Chapel Hill, NC (United States)
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; antibody engineering; epigenetics; antibody validation; protein–protein interaction; data reproducibility