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

Takamitsu Hattori,Shohei Koide,Darson Lai,Irina S Dementieva,Kalina M Świst-Rosowska,Brian D Strahl,Kohei Kurosawa,Louesa R Akin,Alexander J Ruthenburg,Adrian T Grzybowski,Yupeng Zheng,Krzysztof Krajewski,Neil L Kelleher,Akiko Koide,Sherwin P Montaño

doi:10.1073/pnas.1522691113

Abstract

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.

Highlights

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
The limited understanding of the molecular mechanisms underlying the recognition of histone posttranslational modification (PTM) has severely limited our ability to apply mechanism-based designs to the generation of recombinant antibodies to a wider range of histone PTMs
Antigen clasping led to recombinant antibodies for histone posttranslational modifications with extraordinarily high specificity, valuable tools for epigenetic research

Summary

Introduction

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. 309M3-B was more sensitive to modifications of adjacent residues than our first recombinant antibody to H3K9me3, termed 309M3-A, which we reported previously [9], indicating that 309M3-B recognizes many features of the H3K9me3 peptides outside the K9me3 site itself (SI Appendix, Fig. S1).

Results

Conclusion