Molecular determinants of factor VIII immunogenicity Moderator Prof. dr. H.C.J. Eikenboom Prof. dr. H.C.J. Eikenboom 1st author / speaker Jan Voorberg PhD Co-authors Not applicable
Conflict of Interest Disclosure Form In accordance with the rules of the Health Care Inspectorate (IGZ) Name: Jan Voorberg PhD Affiliation: Sanquin Research / University of Amsterdam ☐ I have no potential conflict of interest to report � � I have the following potential conflict(s) of interest to report I have the following potential conflict(s) of interest to report Type of affiliation / financial interest Name of commercial company Receipt of grants/research supports: NOVO Nordisk Research grant. Receipt of honoraria or consultation fees: Participation in a company sponsored speaker’s bureau: Stock shareholder: Other support (please specify): Scientific advisory board
Molecular determinants of factor VIII immunogenicity A major side effect of current treatment of A2 hemophilia A comprises the development of inhibitory antibodies that preclude further treatment with factor VIII treatment with factor VIII A1 A3 Factor VIII inhibitors occur in about 10- 40% of the patients with severe C2 hemophilia A C1
Inhibitor development in a patient with mild hemophilia A FVIII rFVIIa 100 BU/ml) 1.00 Inhibitor titre (BU/ 0.80 10 0.60 0.40 1 0.20 0 0.00 0 50 100 150 200 250 300 Time (days) Van den Brink et al., 1999
FVIII inhibitors are diagnosed using a classical “mixing” assay (Nijmegen Bethesda assay) “Nijmegen modification” of the Bethesda assay can specifically detect low levels of FVIII inhibitors in plasma (<1.0 BU/ml). Verbruggen et al., 1995
Subclass analysis of factor VIII inhibitors reveals a prominent contribution of IgG1 and IgG4 IgG1 IgG4 92 kDa Fulcher et al., 1987
Major B cell epitopes reside in the A2, A3 and C2 domain of factor VIII FX FXa A2 Heavy chain A1 A2 A3 A3 FVIIIa FVIIIa FIXa FIXa A1 A1 C2 C1 phospholipids Light chain C1 C2 Voorberg and Meems, 2014
Summary factor VIII inhibitors (I) • Anti-factor VIII antibodies are primarily composed of subclasses IgG1 and IgG4 • • The majority of anti-factor VIII antibodies bind to the A2, A3 or C2 The majority of anti-factor VIII antibodies bind to the A2, A3 or C2 domain • Anti-A2 and A3 domain antibodies prohibit the binding of factor VIII to factor IXa. Anti-C2 domain antibodies interfere with binding of factor VIII to phospholipids.
FVIII-derived peptides presented on MHC class II FVIII is endocytosed by antigen- presenting cells and loaded on MHC class II for efficient presentation to CD4 + T cells: 1. Endocytosis FVIII 2. Proteolytic degradation 2. Proteolytic degradation 3. Loading small peptides on MHC class II 4. Recognition by FVIII-specific CD4 + T cells Studies using either patient-derived material of mouse models of hemophilia A gave us insights into repertoire of FVIII-derived peptides presented to the T cells: mass spectrometry, humanized mouse models and bioinformatic tools
Peptide presentation on MHC class II employing factor VIII pulsed dendritic cells FVIII PBS MHCII MHCII Van Haren et al., 2011
Broad repertoire of FVIII-specific peptides can be presented on MHC class II Van Haren et al., 2011 ; 2013
Immune recognition of FVIII by antigen-presenting cells A2 A1 A3 C2 C1 Van Haren et al., 2012
Various pathways of endocytosis Parton, R. G. & Simons, K. The multiple faces of caveolae. Nature Reviews Molecular Cell Biology 8, 185–194 (2007) doi:10.1038/nrm2122
FVIII endocytosis is mediated via the C1 domain Effect of antibodies on FVIII �� endocytosis 100 trol % control 75 75 �� VK34 (A2) 50 �� KM33 (C1) 25 0 0 10 40 160 nM antibody added �� �� Van Haren et al., 2012
Modification of an exposed loop in the C1 domain reduces endocytosis of factor VIII WT 100 A2 2092/93 80 2090/92/93 C1 domain A1 A1 % MFI A3 60 40 C2 20 C1 0 R2090 0 10 20 30 40 K2092 FVIII (nM) F2093 Wroblewska et al., 2012
Modification of an exposed loop in the C1 domain reduces inhibitor titers in mouse model of hemophilia A A2 C1 domain A1 A3 * (AU/ml) 750 Cag 9 anti-FVIII IgG (AU/ 1 AU=1 ng Cag C2 C2 C1 C1 600 450 R2090 K2092 300 F2093 150 0 Days: 0 7 14 21 WT 2090/92/93 28 FVIII -/- FVIII Wroblewska et al., 2012
Modification of an exposed loop in the C1 domain reduces CD4+ T cell responses in mouse model of hemophilia A Con A 8 60 Wild-type n (SI) Proliferation (S Proliferation (SI) (SI) 6 6 40 4 *** ** ** 2090/92/93 2 20 0 0.0 0.2 0.4 0.6 0.8 1.0 0 WT 2090/92/93 FVIII (ug/ml) Blood 2012 Wroblewska et al., 2012
Summary factor VIII inhibitors (II) • A broad repertoire of factor VIII derived peptides is presented on MHC class II • • Endocytosis of factor VIII by antigen presenting cells is mediated by Endocytosis of factor VIII by antigen presenting cells is mediated by an exposed loop in the C1 domain of factor VIII
Risk factors for inhibitor development in hemophilia A A2 patient IgG1,4 A1 A1 Immune system Immune system A3 B cells antigen T cells presentation C2 C1
Intron 22; approximately 20% of patients develop inhibitors
Specific missense mutations in patients with mild hemophilia A are linked to inhibitor development Eckhardt et al., 2013
Risk factors for inhibitor development in hemophilia A A2 patient IgG1,4 A1 A1 Immune system Immune system A3 B cells antigen T cells presentation C2 C1 FVIII genotype IL-10, TNFα, CTLA4 polymorphism, GWAS- study Intensity of treatment
Treatment-related factors; FVIII products Gouw et al., 2013
Novel generation of factor VIII products; extended half-life (1.5-1.8) Bay-9027 Studies performed in previously treated patients; no inhibitors patients; no inhibitors PEG FVIII-Fc Blood 2014; JTH 2014
Genetic and treatment-related parameters in a threshold model for inhibitor formation in hemophilia A Gouw and Fijnvandraat et al., 2013; modified from Van Helden et al., 2010.
Acknowledgements Simon van Haren Eszter Herzcenik Aleksandra Wroblewska Sander Meijer Koen Mertens Koen Mertens Jan Voorberg Eddie James Kate Pratt Anja ten Brinke Marijke van den Berg Karin Fijnvandraat
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