Epitope mapping of inhibitory antibodies targeting the C2 domain of coagulation factor VIII by hydrogen–deuterium exchange mass spectrometry

Authors

  • A. M. Sevy,

    1. Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
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  • J. F. Healey,

    1. Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
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  • W. Deng,

    1. Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
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  • P. C. Spiegel,

    1. Department of Chemistry, Western Washington University, Bellingham, WA, USA
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  • S. L. Meeks,

    Corresponding author
    1. Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
    • Correspondence: Shannon Meeks and Renhao Li, Department of Pediatrics, Emory University School of Medicine, 2015 Uppergate Drive NE, Atlanta, GA 30322, USA.

      Tel.: +1 404 727 1608; fax: +1 404 727 4859.

      E-mail: Shannon.meeks@choa.org and

      Tel.: +1 404 727 8217; fax: +1 404 727 4859.

      E-mail: renhao.li@emory.edu

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  • R. Li

    Corresponding author
    1. Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
    • Correspondence: Shannon Meeks and Renhao Li, Department of Pediatrics, Emory University School of Medicine, 2015 Uppergate Drive NE, Atlanta, GA 30322, USA.

      Tel.: +1 404 727 1608; fax: +1 404 727 4859.

      E-mail: Shannon.meeks@choa.org and

      Tel.: +1 404 727 8217; fax: +1 404 727 4859.

      E-mail: renhao.li@emory.edu

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  • Manuscript handled by: R. Camire
  • Final decision: P. H. Reitsma, 17 October 2013

Summary

Background

The development of anti-factor VIII antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A, leading to significant increases in morbidity and treatment cost. Using a panel of mAbs against different epitopes on FVIII, we have recently shown that epitope specificity, inhibitor kinetics and time to maximum inhibition are more important than inhibitor titer in predicting responses to FVIII and the combination of FVIII and recombinant FVIIa. In particular, a subset of high-titer inhibitors responded to high-dose FVIII, which would not be predicted on the basis of their inhibitor titer alone. Thus, the ability to quickly map the epitope spectrum of patient plasma with a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients.

Objectives

To map the epitopes of anti-FVIII mAbs, three of which are classic inhibitors and one of which is a non-classic inhibitor, by the use of hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS).

Methods

The binding epitopes of four mAbs targeting the FVIII C2 domain were mapped with HDX-MS.

Results

The epitopes determined with HDX-MS are consistent with those obtained earlier through structural characterization and antibody competition assays. In addition, classic and non-classic inhibitor epitopes could be distinguished by the use of a limited subset of C2 domain-derived peptic fragments.

Conclusion

Our results demonstrate the effectiveness and robustness of the HDX-MS method for epitope mapping, and suggest a potential role of rapid mapping of FVIII inhibitor epitopes in facilitating individualized treatment of inhibitor patients.

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