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

 

Introduction 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). 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.

Method A database of FVIII peptides presented on different MHC class II alleles was assembled and compared to predicted MHC class II presented epitopes using the ‘Immune Epitope Database (IEDB)’ website. An additional 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 FVIII peptides presented on HLA-DP4, since these alleles are highly prevalent in the Caucasian population.

Results The IEDB website identified a large number of FVIII core peptides presented by multiple MHC class II alleles. To supplement this data-set 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-associated peptides were identified. We detected over 100 HLA-DR and 7 HLA-DP4 presented FVIII peptides. An HLA-DR presented FVIII peptide was fused to a cell-penetrating peptide and incubated with RBCs. FACS analysis revealed its internalization in a dose-dependent manner. FVIII peptide-treated RBCs phagocytosis by macrophages was assessed using microscopy and MHC class II presentation of FVIII-derived peptides on macrophages was validated. We are currently exploring whether the presentation of immunodominant FVIII peptides on MHC class II results in tolerance induction.

Conclusion 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.

Publication History

Article published online:
20 February 2023

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