Factor VIII Proteins Having a Rationally Modified, Immunodominant T-Cell Epitope Demonstrate Normal Procoagulant Activity, Bind To VWF With High Affinity, and Are Markedly Less Stimulatory To FVIII-Specific Human T Cells

Neutralizing anti-factor VIII (FVIII) antibodies, referred to clinically as “inhibitors”, can develop as an alloimmune response in hemophilia A patients receiving FVIII infusions as replacement therapy. Immune Tolerance Induction, consisting of intensive FVIII treatment in an effort to tolerize patients to FVIII, is extraordinarily expensive and not always successful. New approaches to avoid inhibitor development, and to treat patients who develop this deleterious immune response, are needed. Initial stages of inhibitor development include FVIII uptake and processing into peptides by antigen-presenting cells such as dendritic cells (DCs), presentation of FVIII peptides on MHC Class II (HLA) receptors on the cell surface, and recognition of HLA-peptide complexes by one or more circulating T cells. Subsequent signaling through immunological synapses between antigen-presenting cells and T-cell receptors causes proliferation of effector T cells, which secrete cytokines promoting anti-FVIII antibody production.

Our laboratory has been identifying T-cell epitopes in FVIII, which are amino acid sequences 11-15 residues long that bind to specific HLA-DR proteins and are in turn recognized by T cells. FVIII 2194-2205 comprises an immunodominant T-cell epitope that binds to HLA-DRB1*01:01. Peptide-MHC binding experiments established that the side chains of amino acid residues F2196, M2199, A2201 and S2204 fit into the HLA-DRB1*01:01 peptide-binding groove, allowing peptides containing this sequence to be presented on the surface of antigen-presenting cells. Fluorescent HLA-DRB1*01:01 tetramers loaded with FVIII_2194-2213 were used to stain and isolate FVIII-specific T cells from 3 hemophilia A subjects who had an HLA-DRB1*01:01 allele. These cells were expanded in culture to generate T-cell clones and polyclonal lines that recognize this sequence and proliferate in response to it. Stimulation of clones with FVIII peptides containing systematic alanine substitutions demonstrated that MHC anchor residues F2196 and M2199 are important for the immunogenicity of this T-cell epitope. The clones were then stimulated with FVIII peptides and recombinant FVIII-C2 domain proteins in which F2196 was changed to a series of other residues. The effect of substitutions at M2199 was examined using the ProPred computer prediction program. Three substitutions that significantly reduced T-cell proliferation (F2196A, F2196L, F2196K) or were predicted to do so (M2199A, M2199W, M2199R) were introduced into recombinant, B-domain-deleted (BDD)-FVIII. The present study tests the hypothesis that less immunogenic FVIII proteins having normal FVIII procoagulant activity can be produced through rational modification of T-cell epitopes. The F2196K and M2199A muteins were expressed at levels similar to wild-type (WT)-BDD-FVIII in BHK-M cells and were purified from serum-free BHK-M cell supernatants. Purified CD14-positive monocytes from individuals with the HLA-DRB1*01:01 allele were differentiated into DCs and used for antigen-presentation assays in which 4 hemophilic T-cell clones and 2 polyclonal lines were added to the DCs and stimulated with FVIII_2194-2213, WT-FVIII, WT-BDD-FVIII and the BDD-FVIII muteins. One clone proliferated weakly in response to both the WT and mutant proteins, while the remaining 3 clones and 2 lines showed markedly less proliferation in response to the BDD-FVIII muteins. BDD-FVIII-2196K and 2199A had specific activities similar to that of WT-BDD-FVIII (by chromogenic and clotting assays), and surface plasmon resonance confirmed that they retained high-affinity binding to von Willebrand factor. These results provide proof-of-principle for the design of less immunogenic FVIII proteins targeted to specific subsets of hemophilia A patients.