Abstract
Neutralizing antibodies (inhibitors) to replacement factor VIII (FVIII, either plasma derived or recombinant) impair the effective management of hemophilia A1. Individuals with hemophilia A due to major deletions of the FVIII gene (F8) lack antigenically cross-reactive material in their plasma (“CRM-negative”), and the prevalence of inhibitors in these individuals may be as high as 90%. Conversely, individuals with hemophilia A caused by F8 missense mutations are CRM-positive, and their overall prevalence of inhibitors is <10% (ref. 2). Individuals with the F8 intron 22 inversion (found in ∼50% of individuals with severe hemophilia A) have been grouped with the former on the basis of their genetic defect and CRM-negative status. However, only ∼20% of these individuals develop inhibitors3. Here we demonstrate that the levels of F8 mRNA and intracellular FVIII protein in B lymphoblastoid cells and liver biopsies from individuals with the intron 22 inversion are comparable to those in healthy controls. These results support the hypothesis that most individuals with the intron 22 inversion are tolerized to FVIII and thus do not develop inhibitors. Furthermore, we developed a new pharmacogenetic algorithm that permits the stratification of inhibitor risk for individuals and subpopulations by predicting the immunogenicity of replacement FVIII using, as input, the number of putative T cell epitopes in the infused protein and the competence of major histocompatibility complex class II molecules to present such epitopes. This algorithm showed statistically significant accuracy in predicting the presence of inhibitors in 25 unrelated individuals with the intron 22 inversion.
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Acknowledgements
We would like to thank P. Matzinger, B. Golding, J. Lozier, D. Levin, M. Carcao, V. Blanchette, G. Rivard and V. La Terza for helpful comments and J. Sauna for help with the graphics. We thank S.V. Ambudkar (US National Cancer Institute) for the use of facilities, M.J. Lenardo and K. Shafer-Weaver (US National Institute of Allergy and Infectious Diseases) for the gift of the purified C2 domain of FVIII and P. Stock (University of California, San Francisco) for liver tissue samples. These samples were obtained under the University of California University-Wide AIDS Research Program (TP-99-SF-001) Committee on Human Research (H8024-23454) and the Solid Organ Transplantation in HIV: Multi-Site Study (AI052748) funded by the US National Institute of Allergy and Infectious Diseases Committee on Human Research (10-01178). Research conducted in the laboratory of Z.E.S. is funded by the modernization of science program of the Center for Biologics Evaluation and Research, US Food and Drug Administration. Research conducted in the laboratory of T.E.H. is funded by grants from the National Heart, Lung and Blood Institute, National Institutes of Health (1RC2-HL101851, HL-71130 and HL-72533), the Bayer Healthcare Corporation, Bayer Hemophilia Awards Program, Baxter Healthcare Corporation and the Clinical Translational Science Institute at the University of Southern California. The work of T.M.P. is fully supported by the Intramural Program of the US National Library of Medicine, NIH.
Disclaimer: The findings and conclusions in this article have not been formally disseminated by the US Food and Drug Administration and should not be construed to represent any Agency determination or policy.
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G.S.P. executed experiments, analyzed results and generated the first draft of the manuscript. C.Y. performed computational and statistical assessments of peptide–MHC-II binding, analyzed the results and helped edit the manuscript. L.M.M.-J. performed the mass spectrometric analysis. S.G. provided the confocal microscopy images and advice on sample preparation. S.A.C. performed the F8 resequencing, J.E.C. and E.K.M. immortalized cells from the male donor with hemophilia A and the healthy male donor subject. N.R. and D.L. performed the Southern blot–based intron 22 inversion assays on patient samples and reported their results. V.S. and C.K.-S. performed the experiments related to the transient expression of F8 constructs in different cell lines. K.R.V. performed bioinformatic analyses in evaluating all raw F8 DNA sequencing data and helped with statistical analysis. T.M.P. analyzed clinical data, provided advice on statistical analysis and helped with editing early drafts of the manuscript. G.F.P. provided the liver samples, was involved with study design and provided advice on clinical aspects of this study. T.E.H. conceptualized the hypothesis, initiated, designed and coordinated the clinical phases of the study, directed different experimental evaluations and statistical analyses, and generated and edited the first and subsequent drafts of the manuscript. Z.E.S. designed and coordinated different aspects of the study, analyzed results, and generated and edited the first and final drafts of the paper. The PATH Study clinical investigators obtained institutional review board or ethics committee approvals and provided the samples and data used in the study in compliance with their institutional rules and regulations for human subject investigations.
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G.F.P. is an employee of Biogen Idec, and T.E.H. is the founder of Haplomics, which owns international patents and international and US patent applications directed to compositions, methods and diagnostics useful for treating subjects with hemophilia A.
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Pandey, G., Yanover, C., Miller-Jenkins, L. et al. Endogenous factor VIII synthesis from the intron 22–inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A. Nat Med 19, 1318–1324 (2013). https://doi.org/10.1038/nm.3270
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DOI: https://doi.org/10.1038/nm.3270
