Sequence-Modified Factor VIII Variants Having Reduced Immunogenicity

Abstract 39

Neutralizing anti-factor VIII (FVIII) antibodies, or “inhibitors”, interfere with FVIII pro-coagulant activity, and persistent inhibitors can result in significant morbidity and mortality in hemophilia A (HA) patients and in individuals who develop autoantibodies to their endogenous FVIII. Inhibitor production follows stimulation of helper T cells by linear amino acid sequences in FVIII corresponding to HLA-restricted T-cell epitopes. An immunodominant HLA-DRB1*01:01-restricted T-cell epitope within a peptide corresponding to FVIII residues 2194–2213 was identified previously using blood samples from two mild HA subjects with hemophilic mutation A2201P. This same immunodominant epitope was found recently in inhibitor subjects with (a) a large F8 gene deletion and b) an F8 nonsense mutation in exon 12. The present study aims to identify amino acid substitutions in FVIII that will neutralize this T-cell epitope while preserving the pro-coagulant activity of the modified FVIII protein. MHC class II - peptide binding assays were carried out using truncated FVIII peptides to determine the shortest sequence with full binding affinity for a recombinant HLA-DR0101 protein, and subsequently using peptides having systematic arginine substitutions at each position to identify the specific residues that confer this binding affinity. The results indicated that FVIII_2194–2205 is the minimal binding epitope and that residues F2196, M2199, A2201 and S2204 interact with the peptide-binding groove of HLA-DR0101. Next, four T-cell clones that all proliferate in response to this epitope but have different T-cell receptors were stimulated with 12 peptides having systematic alanine substitutions at each position of FVIII_2194–2205. The F2196A substitution abrogated proliferation of all four clones. The M2199A-substituted peptide stimulated three of the clones more weakly than the wild-type peptide. Peptide binding and T-cell assays were next carried out with FVIII_2194–2205 peptides in which the 19 common non-phenylalanine amino acids were substituted at position 2196. These results identified 12 different amino acid substitutions that decreased both MHC binding and T-cell proliferation more than 10-fold. The binding of FVIII_2194–2205 and FVIII_{2194–2205, F2196A} to 10 common HLA-DRB1 proteins was measured to determine the potential promiscuity of this epitope. Moderate or low affinity binding of FVIII_2194–2205 (IC₅₀ < 50 mM) to DR0401, DR0404, DR0901, DR1001, and DR1501 was observed. FVIII_{2194–2205, 2196A} did not bind to any of the HLA-DRB1 proteins, suggesting that this substitution would not introduce a neo-epitope recognized by these other common MHC class II receptors. A recombinant FVIII-C2 domain protein with substitution F2196A was generated in E. coli and purified to homogeneity following a procedure that removes endotoxin. This FVIII-C2 mutein failed to stimulate the same four T-cell clones, all of which showed a strong, dose-dependent response to wild-type FVIII-C2. Recombinant B-domain-deleted FVIII (BDD-FVIII) proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W and M2199R were expressed in BHK-M cell lines. Multiple cell lines were generated to express wild-type BDD-FVIII and each of these mutant proteins. Expression levels of the muteins were similar to that of wild-type BDD-FVIII except for the M2199W and F2196A variants, which had expression levels ∼30 and 10% that of wild-type BDD-FVIII, respectively. Specific activities of the muteins, measured using chromogenic and clotting assays, were similar to that of wild-type BDD-FVIII. Binding of these muteins to plasma-derived von Willebrand factor was evaluated by ELISAs, as a surrogate assay to indicate possible effects of specific mutations on FVIII half-life in the circulation. Their affinities for VWF ranged from ∼40–100% that of wild-type BDD-FVIII. Our results suggest that FVIII muteins with amino acid substitutions that abolish binding to DR0101 and retain reasonable FVIII functionality could be developed as less immunogenic therapeutic proteins, in order to avoid HLA-DRB1*01:01-restricted immune responses in HA patients with this common allele. The immunogenicity of this T-cell epitope and of the sequence-modified peptides and proteins in HA subjects with other HLA-DRB1 alleles is currently under investigation.