PB0185 - Exploring red blood cells as a novel tolerogenic approach for factor VIII inhibitors employing immuno-dominant FVIII derived peptides presented on MHC class II

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

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

MD PhD
Department of Pediatric Hematology, Amsterdam UMC, University of Amsterdam, Emma Children’s Hospital, Amsterdam, The 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 biotherapeutics immunogenicity. Here we employed bioinformatics and advanced 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 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 that were shown to be presented by multiple MHC class II alleles. To supplement this data-set we successfully developed a 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 5 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. We are currently exploring whether FVIII peptide-treated RBCs phagocytosis by macrophages results in tolerogenic presentation of immunodominant FVIII peptides on MHC class II.

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.