Identification and removal of a promiscuous CD4+T cell epitope from the C1 domain of factor VIII

Background: The development of inhibitors in hemophiliacs is a severe complication of factor VIII (FVIII) replacement therapy and is a process driven by FVIII specific T helper cells. Objectives: To finely map T cell epitopes within the whole FVIII protein in order to investigate the possibility of engineering FVIII variants with reduced propensity for inhibitor development. Patients and methods: T cell lines were generated from five patients with severe hemophilia who had developed inhibitors, and were screened for T cell proliferation against pools of overlapping peptides spanning the entire B domain deleted (BDD) FVIII sequence. Positive peptide pools were decoded by screening individual peptides against the T cell lines. Positive peptides, and mutants thereof, were tested for their ability to bind major histocompatibility complex (MHC) Class II and stimulate T cell proliferation in a panel of healthy donors. The activities of the corresponding mutant proteins were assessed via chromogenic assay. Results: One peptide, spanning FVIII amino acids 2098-2112, elicited a vigorous response from one hemophiliac donor, induced strong T cell responses in the panel of healthy donors and bound to a number of HLA-DR alleles. Mutations were made in this peptide that removed its ability to stimulate T cells of healthy donors and to bind to MHC Class II while retaining full activity when incorporated into a mutant BDD-FVIII protein. Conclusions: Fine T cell epitope mapping of the entire FVIII protein is feasible, although challenging, and this knowledge may be used to create FVIII variants which potentially have reduced immunogenicity.