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    Oral Communication

    Hemophilia and Rare Bleeding Disorders

    Hemophilia Basic Science - Inhibitors

    OC 44.3 - Exploring red blood cells as a novel tolerogenic approach for factor VIII inhibitors employing immuno-dominant FVIII derived peptides presented on MHC class II

    Monday, June 26, 2023
    15:15 – 15:30 ET
    Room: 520
    Session Available As: On-Demand

      Presenting Author(s)

    • Mariarosaria Miranda, MA

      PhD student
      Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
      Amsterdam, Noord-Holland, Netherlands

      • Co-Author(s)

    • EB

      Eelke Brandsma

      PostDoc
      Department of Hematopoiesise, Sanquin Research, Amsterdam, The Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • PK

      Paul Kaijen

      Technician
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • FV

      Floris Van Alphen

      Technician
      Department of Molecular Hematology, Sanquin Research, 1066 CX Amsterdam, the Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • RV

      Robin Van Bruggen

      PI
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • KF

      Karin Fijnvandraat

      Professor
      Amsterdam UMC, University of Amsterdam, Emma Children’s Hospital, Pediatric Hematology Amsterdam, Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • Sebastien Lacroix-Desmazes, PhD

      PI
      Centre De Recherche Des Cordeliers, Paris, France
      Paris, Ile-de-France, France

    • JV

      Jan Voorberg

      Professor, Principal investigator
      Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory
      Amsterdam, Noord-Holland, Netherlands

    Background: The main complication of hemophilia A treatment is the development of neutralizing antibodies (inhibitors) against factor VIII (FVIII). The eradication of anti-FVIII antibodies relies on immune tolerance induction (ITI).

    Aims: Since ITI is efficient in only 60-80% of cases, within the EDUC8-consortium we are developing innovative methods to reduce the immunogenicity of biotherapeutics. Here we employed bioinformatics and proteomic-based peptide presentation assays to identify promiscuously presented FVIII peptides to use in pioneering immuno-tolerogenic approaches. To this end, we are exploring red blood cells (RBCs) as innovative antigen delivery system to induce tolerance.

    Methods: A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocytes-derived DCs from HLA-typed healthy donors. Specific attention was on the identification of HLA-DP4-FVIII derived peptides, since these alleles are highly prevalent in the Caucasian population.

    Results: We successfully developed a mass-spectrometry based protocol to study HLA-DR and HLA-DP antigen presentation utilizing specific monoclonal antibodies. Using this novel approach, approximately 4000 HLA-DR and 2000 HLA-DP presented peptides were identified. We detected over 100 HLA-DR and 10 HLA-DP4 presented FVIII peptides (Table 1). Three HLA-DR presented FVIII peptide were fused to a cell-penetrating peptide and incubated with RBCs. Flow cytometry revealed their binding to RBCs in a dose-dependent manner. Binding of FVIII derived peptides to RBCs was confirmed by ImageStream analysis (Figure 1). FVIII peptide-treated RBCs were efficiently endocytosed by macrophages. Functional presentation of RBC bound peptides on MHC class II on the surface of macrophages was established employing immunopeptidomic approaches. We are currently exploring whether the presentation of immunodominant FVIII peptides on MHC class II results in tolerance induction.

    Conclusion(s): Our data provide an inventory of promiscuously presented FVIII-derived peptides which will guide the development of novel tolerogenic approaches for FVIII inhibitors employing RBCs as carrier.

    Learning Objectives:

    • discuss the optimal approach to delineate HLA class II FVIII peptidome and its relevance in addressing the immunogenicity of FVIII.
    • explain how Red Blood Cells can be involved in the tolerance induction
    • examine the main compliance in hemophilia A, explore promising strategies for monitoring FVIII immunogenicity, and developing novel approaches to induce tolerance