Case study: Specification of CD4+ T cell epitopes of human FVIII. Birgit Reipert Director Immunology TA Hemophilia/Hematology Baxter BioScience

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1 Case study: Specification of CD4+ T cell epitopes of human FVIII Birgit Reipert Director Immunology TA Hemophilia/Hematology Baxter BioScience Mastering Immunogenicity, Boston MA, September

2 Hemophilia A X-linked recessive bleeding disorder that affects 1 in 5,000-10,000 men caused by mutations in the gene that codes for human clotting factor VIII gene mutations lead to either diminished function of factor VIII or lack of endogenous production of factor VIII Clinic: spontanous bleedings and hemorrhages 2

3 Major complication of replacement therapy in hemophilia A Replacement therapy with factor VIII factor VIII? ~30% patients develop neutralizing anti-factor VIII antibodies (factor VIII inhibitors) patients with severe hemophilia A Scharrer I et al. Haemophilia. 1999;5: no factor VIII inhibitors 3

4 T-cell dependent antibody responses against proteins BCR: B cell receptor TCR: T cell receptor protein BCR co-stimulation peptides MHCclassII activated B cell TCR activated CD4 + T cell Y antibodies plasma cell 4

5 T-cell dependent antibody responses against proteins BCR: B cell receptor TCR: T cell receptor dendritic cell protein protein co-stimulation MHCclassII TCR BCR co-stimulation peptides peptides MHCclassII activated B cell TCR activated CD4 + T cell CD4 + T cell Y antibodies plasma cell 5

6 T-cell dependent antibody responses against proteins BCR: B cell receptor TCR: T cell receptor dendritic cell protein protein co-stimulation MHCclassII TCR BCR co-stimulation peptides peptides MHCclassII activated B cell TCR activated CD4 + T cell CD4 + T cell Y antibodies plasma cell 6

7 Flexibility and plasticity of peripheral CD4 + T cells dendritic cell O Shea et al. Science. 2010; 327(5969):

8 Flexibility and plasticity of peripheral CD4 + T cells induction of antibodies dendritic cell induction of immune tolerance O Shea et al. Science. 2010; 327(5969):

9 Foxp3+ CD4 + T cells favour induction of immune tolerance What favours the induction of tolerizing Foxp3 + Tregs (regulatory T cells)? chronic exposure to antigen exposure to antigen in the absence of pro-inflammatory stimuli/danger signals.. dendritic cell O Shea et al. Science. 2010; 327(5969):

10 Specification of CD4 + T cell epitopes of human factor VIII 10

11 T-cell dependent antibody responses against proteins B cells and T cells recognize proteins in different ways Antigen-presenting cell B-cell receptor (BCR) protein T-cell receptor (TCR) MHC-class II peptide B cell CD4 + T cell 11

12 Peptides presented by MHC-class II T-cell receptor contact residues peptide P1 P4 P6 P9 MHC-class II anchor residues of the peptide α1 α2 β1 β2 MHC-class II molecule Mastering Immunogenicity, Boston MA, September

13 Specification of CD4 + T cell epitopes of human factor VIII Our Approaches: 1. Humanized hemophilic mice 2. In vitro binding assays using the Pro Immune REVEAL MHC peptide binding assay Mastering Immunogenicity, Boston MA, September

14 Partially humanized hemophilic mice for identification of major factor VIII T-cell epitopes E17 hemophilia A mouse (kockout of the murine factor VIII gene - ) that carries human HLA-DRB1*1501 ( ) and does not express any murine MHC-class II ( ) 14

15 Partially humanized hemophilic mice for identification of major factor VIII T-cell epitopes E17 hemophilia A mouse (kockout of the murine factor VIII gene - ) that carries human HLA-DRB1*1501 ( ) and does not express any murine MHC-class II ( ) Induction of antibody response against proteins depends on the presentation of immunogenic peptides by the human HLA-DRB1*

16 Partially humanized hemophilic mice for identification of major factor VIII T-cell epitopes E17 hemophilia A mouse (kockout of the murine factor VIII gene - ) that carries human HLA-DRB1*1501 ( ) and does not express any murine MHC-class II ( ) Induction of antibody response against proteins depends on the presentation of immunogenic peptides by the human HLA-DRB1*1501 The MHC-class II haplotype HLA-DRB1*1501 is associated with an increased risk for patients to develop neutralizing antibodies against factor VIII (Oldenburg et al. 1997; Pavlova et al. 2009) 16

17 CD4 + T-cell responses specific for factor VIII factor VIII (1,000 ng/dose) weeks blood draw i.v. s.c. human MHC-classII i.v. application human MHC-classII s.c. application 17

18 CD4 + T-cell responses specific for factor VIII factor VIII (1,000 ng/dose) weeks i.v. blood draw What are the major factor VIII T-cell epitopes (factor VIII peptides presented by MHC-classII) that drive immune response after i.v. and after s.c. factor VIII? s.c. human MHC-classII i.v. application human MHC-classII s.c. application 18

19 CD4 + T-cell responses specific for factor VIII factor VIII i.v. or s.c. in weekly intervals spleen spleen cells in vitro re-stimulation with factor VIII for 3 days fusion with BW thymoma cells (don`t express T cell receptor) master cell bank/ working cell bank screening of factor VIII peptide library cloning and subcloning selection of factor VIII-specific hybridomas T cell hybridomas (carry T cell receptor of the original splenic T cell) 19

20 CD4 + T-cell responses specific for factor VIII 1 Human factor VIII 2332 A1 a1 A2 a2 B a3 A3 C1 C2 peptide library (15 mers with 3 AA offset) 780 different peptides consisting of 15 amino acids each were tested in pools Each T-cell hybridoma was tested with the whole peptide library to specify the T-cell epitope that is recognized 20

21 CD4 + T-cell responses specific for factor VIII factor VIII (1,000 ng/dose) i.v. factor VIII - 62 hybridomas tested so far weeks blood draw i.v. s.c. human MHC-classII i.v. application human MHC-classII s.c. application 21

22 CD4 + T-cell responses specific for factor VIII factor VIII (1,000 ng/dose) i.v. factor VIII - 62 hybridomas tested so far weeks blood draw i.v. s.c. factor VIII hybridomas tested so far s.c. human MHC-classII i.v. application human MHC-classII s.c. application 22

23 Validation of T-cell epitopes factor VIII i.v. or s.c. in weekly intervals spleen spleen cells restimulation with factor VIII or peptides for 6 hours FACS analysis of activated CD4 + T cells after staining of spleen cells with: live/dead cell marker anti-cd4 antibody anti-cd154 (CD40L) antibody (activation marker, intracellular staining) 23

24 Validation of T-cell epitopes 24

25 CD154 (CD40L) Validation of T-cell epitopes medium control 0.02% 0.16% factor VIII CD4 25

26 CD154 (CD40L) Validation of T-cell epitopes medium control factor VIII U, W, X (mix of 3 major peptides) scu, scw, scx (mix of scrambled peptides) 0.02% 0.16% 0.14% 0.02% CD4 26

27 CD154 (CD40L) Validation of T-cell epitopes medium control factor VIII U, W, X (mix of 3 major peptides) scu, scw, scx (mix of scrambled peptides) 0.02% 0.16% 0.14% 0.02% 0.08% peptide U 0.07% peptide W 0.08% peptide X CD4 27

28 Pro Immune REVEAL MHC peptide binding assay The assay determines the ability of each candidate peptide to bind to a MHC-class II protein of choice compared to a positive and an intermediate control peptide The assay is measuring the ability of each peptide to stabilize the MHC-peptide complex. Detection is based on the presence or absence of the native conformation of this MHC-peptide complex. Birgit Reipert HRSU Barcelona, March 25th

29 Binding of factor VIII peptides to multiple MHC-class II haplotypes DRA*0101 DRB1*1501 DRA*0101;DRB1*0701 DRA*0101;DRB1*0301 DRA*0101;DRB1*1101 DRA*0101;DRB1*0101 DRA*0101;DRB1*1301 DRA*0101;DRB1*0401 US Europe World 10.6% 11.5% 6.2% 11.8% 11.6% 8.3% 10.2% 15.3% 6.5% 5.6% 6.1% 4.1% 7.0% 7.5% 3.1% 5.7% 6.3% 3.1% 6.6% 6.4% 1.7% 29

30 Most of the factor VIII peptides identified bind to multiple MHC-class II haplotypes FVIII CD4 + T-cell epitopes identified In humanized hemophilic HLA-DRB1*1501 mice Cumulative Pan-Allele ProImmune REVEAL TM Score DRA*0101 DRB1*1501 R S T U W X Y Z DRA*0101;DRB1*0701 DRA*0101;DRB1*0301 DRA*0101;DRB1*1101 DRA*0101;DRB1*0101 DRA*0101;DRB1*1301 DRA*0101;DRB1*0401 US Europe World 10.6% 11.5% 6.2% 11.8% 11.6% 8.3% 10.2% 15.3% 6.5% 5.6% 6.1% 4.1% 7.0% 7.5% 3.1% 5.7% 6.3% 3.1% 6.6% 6.4% 1.7% 30

31 Most of the factor VIII peptides identified bind to multiple MHC-class II haplotypes FVIII CD4 + T-cell epitopes identified In humanized hemophilic HLA-DRB1*1501 mice Cumulative Pan-Allele ProImmune REVEAL TM Score DRA*0101 DRB1*1501 R S T U W X Y Z DRA*0101;DRB1*0701 DRA*0101;DRB1*0301 DRA*0101;DRB1*1101 DRA*0101;DRB1*0101 DRA*0101;DRB1*1301 DRA*0101;DRB1*0401 US Europe World 10.6% 11.5% 6.2% 11.8% 11.6% 8.3% 10.2% 15.3% 6.5% 5.6% 6.1% 4.1% 7.0% 7.5% 3.1% 5.7% 6.3% 3.1% 6.6% 6.4% 1.7% 31

32 Conclusion Antibody responses against factor VIII in humanized hemophilic HLA-DRB1*1501 mice depend on the application route. The incidence of antibodies is higher after s.c. than after i.v. application. A limited set of factor VIII peptides (CD4 + T-cell epitopes) drive anti-factor VIII immune responses in humanized hemophilic HLA-DRB1*1501 mice. Immunodominant factor VIII peptides are the same after i.v. and s.c. application of factor VIII. Factor VIII-specific CD4 + T cell epitopes identified in humanized hemophilic HLA-DRB1*1501 mice bind to a number of different HLA-DRB1* haplotypes when tested with ProImmune`s REVEAL class II technology. These results indicate that most of the factor VIII epitopes identified are promiscuous epitopes. 32

33 Acknowledgement Katharina Steinitz PhD student Baxter BioScience Lars Fugger Oxford University UK Brigitte Binder Research Associate Baxter BioScience Center for Innovation and Technology City of Vienna, Austria David Wraith Bristol University UK 33

34 Thank you for your attention 34

35 neutralizing anti-fviii antbodies (BU/ml) Antibody responses against FVIII 8 i.v. doses of FVIII (1000ng), given in weekly intervals E17 hemophilic mice murine MHC-class II 10 1 E17 hemophilic mice human MHC-class II E17 hemophilic mice no MHC-class II ELISA titer of anti-fviii antbodies 35