Molecular determinants of factor VIII immunogenicity Moderator - - PowerPoint PPT Presentation

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

• 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

I have the following potential conflict(s) of interest to report

Molecular determinants of factor VIII immunogenicity

A major side effect of current treatment of hemophilia A comprises the development of inhibitory antibodies that preclude further treatment with factor VIII

A2

treatment with factor VIII Factor VIII inhibitors occur in about 10- 40% of the patients with severe hemophilia A

A1 C2 C1 A3

100 1.00

FVIII rFVIIa

BU/ml)

Inhibitor development in a patient with mild hemophilia A

Time (days)

50 100 150 200 250 300 1 10 0.00 0.20 0.40 0.60 0.80

Inhibitor titre (BU/

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 Fulcher et al., 1987 92 kDa

Major B cell epitopes reside in the A2, A3 and C2 domain of factor VIII

A2 A1 A3

Heavy chain

A1 A2 A3 FIXa FX FXa FVIIIa

A1 C1 C2

Light chain

C1

FIXa

C2

phospholipids

FVIIIa

Voorberg and Meems, 2014

• 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

Summary factor VIII inhibitors (I)

• 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 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

FVIII-derived peptides presented on MHC class II

• 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 C2 C1 A3

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

75 100

trol

• FVIII endocytosis is mediated via the C1 domain

Effect of antibodies on FVIII endocytosis

10 40 160 25 50 75

KM33 (C1) VK34 (A2)

nM antibody added % control

• Van Haren et al., 2012

C1 domain A2 A1

Modification of an exposed loop in the C1 domain reduces endocytosis of factor VIII

80 100

WT 2092/93 2090/92/93

R2090 F2093 K2092

A3 A1 C1 C2

10 20 30 40

20 40 60

FVIII (nM)

% MFI

Wroblewska et al., 2012

750

*

(AU/ml) Cag 9

C1 domain A2 A3 A1 C1 C2

Modification of an exposed loop in the C1 domain reduces inhibitor titers in mouse model of hemophilia A

WT 2090/92/93

150 300 450 600

anti-FVIII IgG (AU/ 1 AU=1 ng Cag

7 14 21 28 Days: FVIII

FVIII-/-

R2090 F2093 K2092

C1 C2

Wroblewska et al., 2012

6 8

Wild-type n (SI)

60

(SI)

Con A

Modification of an exposed loop in the C1 domain reduces CD4+ T cell responses in mouse model of hemophilia A

0.0 0.2 0.4 0.6 0.8 1.0 2 4 6

** ** ***

2090/92/93 FVIII (ug/ml) Proliferation (S

WT 2090/92/93 20 40

Proliferation (SI)

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 A1

Immune system IgG1,4 patient

A1 C2 C1 A3

Immune system B cells T cells antigen presentation

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 A1

Immune system IgG1,4 patient

A1 C2 C1 A3

Immune system B cells T cells antigen presentation

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

PEG

Blood 2014; JTH 2014

FVIII-Fc

patients; no inhibitors

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